Chondroitin polymerase and DNA encoding the same

ABSTRACT

A chondroitin polymerase having such properties that it transfers GlcUA and GalNAc alternately to a non-reduced terminal of a sugar chain from a GlcUA donor and a GalNAc donor, respectively, and the like; and a process for producing the chondroitin polymerase.

BACKGROUND OF THE PRESENT INVENTION

[0001] 1. Field of the Present Invention

[0002] The present invention relates to a novel chondroitin polymerase(chondroitin synthase), a DNA encoding the same, a method for producingthe chondroitin polymerase, a method for producing a sugar chain havingthe disaccharide repeating unit of chondroitin, a hybridization probefor the chondroitin polymerase and the like.

[0003] 2. Brief Description of the Background Art

[0004] First, abbreviations commonly used in the present specificationare described.

[0005] In the formulae and the like, “GlcUA”, “GalNAc”, “GlcNAc”, “UDP”and “-” represent D-glucuronic acid, N-acetyl-D-galactosamine,N-acetyl-D-glucosamine, uridine 5′-diphosphate and a glycosidic linkage,respectively.

[0006] Chondroitin is a sugar chain comprised of a disacchariderepeating structure of GlcUA residue and GalNAc residue(-GlcUAβ(1-3)-GalNAcβ(1-4)-; hereinafter also referred to as“chondroitin backbone”), and a sugar chain in which the chondroitin isfurther sulfated is chondroitin sulfate.

[0007] Regarding an enzyme which synthesizes chondroitin from a GlcUAdonor and a GalNAc donor by alternately transferring GlcUA and GalNAc toan acceptor (chondroitin polymerase or chondroitin synthase) and DNAwhich encodes the same, only a Pasteurella multocida chondroitinsynthase (J. Biol. Chem., 275(31), 24124-24129 (2000)) is known.

[0008] Also, certain Escherichia coli strain (Escherichia coli serotype05:K4(L):H4, hereinafter referred to as “Escherichia coli strain K4”)produces a polysaccharide having a chondroitin backbone, as a capsularantigen, but its structure is a trisaccharide repeating structure inwhich fructose is linked to a side chain of the GlcUA residue at theβ2-3 position. Accordingly, it was unclear whether the Escherichia colistrain K4 really has a chondroitin polymerase as its own capsularantigen synthesizing system.

SUMMARY OF THE INVENTION

[0009] An object of the present invention is to provide a novelchondroitin polymerase, a DNA encoding the same, a process for producingthe chondroitin polymerase, a process for producing a sugar chain havingthe disaccharide repeating unit of chondroitin, a hybridization probefor the chondroitin polymerase and the like.

[0010] This and other objects of the present invention have beenaccomplished by a novel chondroitin polymerase having specificproperties described below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 shows a restriction map of λ phage clones which contain apart of R-I region or R-III region of the K antigen gene cluster ofEscherichia coli strain K4.

[0012]FIG. 2 shows the open reading frame (ORF) of the R-II region ofthe K antigen gene cluster of Escherichia coli strain K4.

[0013]FIG. 3 is a graph showing transfer of GalNAc to hexasaccharide ofchondroitin sulfate C by the enzyme of the present invention.

[0014]FIG. 4 is a graph showing transfer of GalNAc to hexasaccharide ofchondroitin sulfate C by the enzyme of the present invention and thesizes of the produced sugar chain.

[0015]FIG. 5 is a graph showing transfer of each monosaccharide whenUDP-GlcUA, UDP-GlcNAc or UDP-GalNAc was used as the donor, andhexasaccharide or heptasaccharide of chondroitin sulfate C was used asthe acceptor.

[0016]FIG. 6 is a graph showing the influence of temperature on theactivity of the enzyme of the present invention.

[0017]FIG. 7 is a graph showing gel filtration patterns of enzymereaction products after various enzyme reaction times.

[0018]FIG. 8 is a graph showing the relationship between the enzymereaction time and the incorporation amount of radioactivity.

[0019]FIG. 9 is a graph showing the relationship between theincorporated radioactivity (V) and the substrate concentration ofUDP-sugar (S).

[0020]FIG. 10 is a graph showing double reciprocal plots.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present inventors have conducted intensive studies and founda novel chondroitin polymerase produced by specific microorganism(Escherichia coli strain K4 (Escherichia coli serotype 05:K4(L):H4, ATCC23502)), isolated cDNA encoding the chondroitin polymerase, andsucceeded in preparing the chondroitin polymerase using the cDNA. Thus,the present invention has been completed.

[0022] Also, this and other objects of the present invention have beenaccomplished by a process for producing the chondroitin polymerase byisolating cDNA encoding the chondroitin polymerase and using the cDNA.The term “chondroitin synthesis” or “synthesis of chondroitin” as usedherein is a concept which includes elongation of the sugar chain ofchondroitin by transferring and adding monosaccharides to a sugar chainsuch as chondroitin. Accordingly, the reaction for elongating the sugarchain of chondroitin by alternately transferring and adding thechondroitin synthesizing monosaccharides (GlcUA and GalNAc) to the sugarchain is included in a concept of “chondroitin synthesis” or “synthesisof chondroitin”.

[0023] The present invention relates to a chondroitin polymerase(hereinafter also referred to as “the enzyme of the present invention”)having the following properties:

[0024] (1) Action:

[0025] the polymerase transfers GlcUA and GalNAc alternately to anon-reduced terminal of a sugar chain from a GlcUA donor and a GalNAcdonor, respectively;

[0026] (2) Substrate Specificity:

[0027] the polymerase transfers GlcUA to an oligosaccharide havingGalNAc on its non-reduced terminal and a chondroitin backbone from aGlcUA donor, but does not substantially transfer GalNAc to theoligosaccharide from a GalNAc donor;

[0028] the polymerase transfers GalNAc to an oligosaccharide havingGlcUA on its non-reduced terminal and a chondroitin backbone from aGalNAc donor, but does not substantially transfer GlcUA to theoligosaccharide from a GlcUA donor;

[0029] (3) Influence by Metal Ions and the Like:

[0030] the polymerase acts in the presence of Mn²⁺ ion but does notsubstantially act in the presence of Ca²⁺ ion, Cu²⁺ ion orethylenediaminetetraacetic acid.

[0031] The enzyme of the present invention is preferably derived fromEscherichia coli.

[0032] The present invention relates to a protein selected from thegroup consisting of the following (A) and (B) (hereinafter also referredto as “the protein of the present invention”):

[0033] (A) a protein comprising the amino acid sequence represented bySEQ ID NO: 2;

[0034] (B) a protein comprising the amino acid sequence in which one ora few amino acid residue(s) in the amino acid sequence represented bySEQ ID NO: 2 are deleted, substituted, inserted or transposed, andhaving a chondroitin polymerase activity.

[0035] The present invention relates to a DNA comprising any one of thefollowing (a) to (c) (hereinafter also referred to as “the DNA of thepresent invention”):

[0036] (a) a DNA which encodes a protein consisting of the amino acidsequence represented by SEQ ID NO: 2;

[0037] (b) a DNA which encodes a protein consisting of an amino acidsequence in which one or a few amino acid residue(s) in the amino acidsequence represented by SEQ ID NO: 2 are deleted, substituted, insertedor transposed, and having a chondroitin polymerase activity;

[0038] (c) a DNA which hybridizes with

[0039] (i) the DNA in (a),

[0040] (ii) a DNA complementary to the DNA in (a), or

[0041] (iii) a DNA having a part of nucleotide sequences of the DNA in(i) and (ii) under stringent conditions.

[0042] The DNA in (a) is preferably represented by SEQ ID NO: 1.

[0043] The present invention relates to a vector comprising the DNA ofthe present invention (hereinafter also referred to as “the vector ofthe present invention”).

[0044] The vector of the present invention is preferably an expressionvector.

[0045] The present invention relates to a transformant in which a hostis transformed with the vector of the present invention (hereinafteralso referred to as “the transformant of the present invention”).

[0046] The present invention relates to a process for producing achondroitin polymerase, which comprises: growing the transformant of thepresent invention; and collecting the chondroitin polymerase from thegrown material (hereinafter also referred to as “the enzyme productionprocess of the present invention”).

[0047] The present invention relates to a sugar chain synthesizingagent, comprising an enzyme protein which comprises an amino acidsequence represented by the following (A) or (B) and has enzymicactivities of the following (i) and (ii) (hereinafter also referred toas “the synthesizing agent of the present invention”):

[0048] (A) the amino acid sequence represented by SEQ ID NO: 2;

[0049] (B) an amino acid sequence in which one or a few amino acidresidue(s) in the amino acid sequence represented by SEQ ID NO: 2 aredeleted, substituted, inserted or transposed;

[0050] (i) GlcUA and GalNAc are alternately transferred to a non-reducedterminal of a sugar chain from a GlcUA donor and a GalNAc donor,respectively;

[0051] (ii) GlcNAc is transferred to a non-reduced terminal of a sugarchain having GlcUA on the non-reduced terminal from a GlcNAc donor.

[0052] The present invention relates to a process for producing a sugarchain represented by the following formula (3), which comprises at leasta step of allowing the synthesizing agent of the present invention tocontact with a GalNAc donor and a sugar chain represented by thefollowing formula (1) (hereinafter also referred to as “the sugar chainproduction process 1 of the present invention”):

GlcUA-X—R¹   (1)

GalNAc-GlcUA-X—R¹   (3)

[0053] wherein X represents GalNAc or GlcNAc; R¹ represents an anygroup; and other symbols have the same meanings as described above.

[0054] The present invention relates to a process for producing a sugarchain represented by the following formula (4), which comprises at leasta step of allowing the synthesizing agent of the present invention tocontact with a GlcNAc donor and a sugar chain represented by thefollowing formula (1) (hereinafter referred to as “the sugar chainproduction process 2 of the present invention”):

GlcUA-X—R¹   (1)

GlcNAc-GlcUA-X—R¹   (4)

[0055] wherein all symbols have the same meanings as described above.

[0056] The present invention relates to a process for producing a sugarchain represented by the following formula (5), which comprises at leasta step of allowing the synthesizing agent of the present invention tocontact with a GlcUA donor and a sugar chain represented by thefollowing formula (2) (hereinafter also referred to as “the sugar chainproduction process 3 of the present invention”):

GalNAc-GlcUA-R²   (2)

GlcUA-GalNAc-GlcUA-R²   (5)

[0057] wherein R² represents an any group; and other symbols have thesame meanings as described above.

[0058] The present invention relates to a process for producing a sugarchain selected from the following formulae (6) and (8), which comprisesat least a step of allowing the synthesizing agent of the presentinvention to contact with a GalNAc donor, a GlcUA donor and a sugarchain represented by the following formula (1) (hereinafter alsoreferred to as “the sugar chain production process 4 of the presentinvention”):

GlcUA-X—R¹   (1)

(GlcUA-GalNAc)n-GlcUA-X—R¹   (6)

GalNAc-(GlcUA-GalNAc)n-GlcUA-X—R¹   (8)

[0059] wherein n is an integer of 1 or more, and other symbols have thesame meanings as described above.

[0060] The present invention relates to a process for producing a sugarchain selected from the following formulae (7) and (9), which comprisesat least a step of allowing the synthesizing agent of the presentinvention to contact with a GalNAc donor, a GlcUA donor and a sugarchain represented by the following formula (2) (hereinafter alsoreferred to as “the sugar chain production process 5 of the presentinvention”):

GalNAc-GlcUA-R²   (2)

(GalNAc-GlcUA)n-GalNAc-GlcUA-R²   (7)

GlcUA-(GalNAc-GlcUA)n-GalNAc-GlcUA-R²   (9)

[0061] wherein all symbols have the same meanings as described above.

[0062] The present invention relates to a hybridization probe comprisinga nucleotide sequence complementary to the nucleotide sequencerepresented by SEQ ID NO: 1 or a part thereof (hereinafter also referredto as “the probe of the present invention”).

[0063] The present invention relates to a glycosyltransfer catalyst(hereinafter also referred to as “the catalyst of the presentinvention”) which comprises an enzyme protein comprising an amino acidsequence selected from the following (A) and (B), and is capable oftransferring GlcUA, GalNAc and GlcNAc to a non-reduced terminal of asugar chain from a GlcUA donor, a GalNAc donor and a GlcNAc donor,respectively:

[0064] (A) the amino acid sequence represented by SEQ ID NO: 2;

[0065] (B) an amino acid sequence in which one or a few amino acidresidue(s) in the amino acid sequence represented by SEQ ID NO: 2 aredeleted, substituted, inserted or transposed.

[0066] The present invention is explained below in more detail.

[0067] <1> Enzyme of the Present Invention and Protein of the PresentInvention

[0068] The enzyme of the present invention is a chondroitin polymerasehaving the following properties (1) to (3).

[0069] (1) Action:

[0070] The enzyme of the present invention transfers GlcUA and GalNAcalternately to a non-reduced terminal of a sugar chain from a GlcUAdonor and a GalNAc donor, respectively.

[0071] As the GlcUA donor, a nucleoside diphosphate-GlcUA is preferred,and UDP-GlcUA is particularly preferred. Also, as the GalNAc donor, anucleoside diphosphate-GalNAc is preferred, and UDP-GalNAc isparticularly preferred.

[0072] The enzyme of the present invention transfers GlcUA and GalNAcalternately to a non-reduced terminal of a sugar chain (acceptor) fromthese respective saccharide donors. For example, when GlcUA is firsttransferred to a non-reduced terminal of a sugar chain (acceptor),monosaccharides are transferred in such a manner that GalNAc is thentransferred, GlcUA is then transferred, GalNAc is then transferred andso on. In the same manner, when GalNAc is first transferred to thenon-reduced terminal of a sugar chain (acceptor), monosaccharides aretransferred in such a manner that GlcUA is then transferred, GalNAc isthen transferred, GlcUA is then transferred and so on. As a result, adisaccharide repeating structure of GlcUA residue and GalNAc residue,namely a chondroitin backbone, is synthesized by the enzyme of thepresent invention.

[0073] As the sugar chain which becomes the acceptor of monosaccharides,a sugar chain having a chondroitin backbone is preferable. As the sugarchain having a chondroitin backbone, chondroitin sulfate and chondroitincan be exemplified. Among chondroitin sulfates, a chondroitin sulfatewhich is mainly comprising a chondroitin 6-sulfate structure and alsocontains a small amount of chondroitin 4-sulfate structure (hereinafterreferred to as “chondroitin sulfate C”) is preferable.

[0074] Also, the sugar chain which becomes an acceptor is morepreferably an oligosaccharide. The size of the oligosaccharide is notparticularly limited, but when the acceptor is an oligosaccharide ofchondroitin sulfate C, hexasaccharide or heptasaccharide is preferable,and tetrasaccharide or hexasaccharide is preferable when it is anoligosaccharide of chondroitin.

[0075] Also, it is preferable that the enzyme of the present inventionis capable of further transferring GalNAc to a sugar chain having ahyaluronic acid backbone (a disaccharide repeating structure of GlcUAresidue and GlcNAc residue) from a GalNAc donor. It is preferable thatthe sugar chain having a hyaluronic acid backbone is also anoligosaccharide. The size of the oligosaccharide is not particularlylimited, but those which are composed of about hexasaccharides areparticularly preferable.

[0076] (2) Substrate Specificity:

[0077] The enzyme of the present invention transfers GlcUA to anoligosaccharide having GalNAc on its non-reduced terminal and achondroitin backbone from a GlcUA donor, but does not substantiallytransfer GalNAc to the oligosaccharide from a GalNAc donor.

[0078] The enzyme of the present invention transfers GalNAc to anoligosaccharide having GlcUA on its non-reduced terminal and achondroitin backbone from a GalNAc donor, but does not substantiallytransfer GlcUA to the oligosaccharide from a GlcUA donor.

[0079] It is preferable that the enzyme of the present invention whichfurther does not substantially transfer GlcNAc from a GlcNAc donor to anoligosaccharide having GalNAc on its non-reduced terminal and also has achondroitin backbone. Furthermore, it is preferable that the enzyme ofthe present invention further transfers GlcNAc from a GlcNAc donor to anoligosaccharide having GlcUA on its non-reduced terminal and also has achondroitin backbone. However, it is preferable that GlcUA is notsubstantially transferred from a GlcUA donor to an oligosaccharideproduced by the transfer of GlcNAc.

[0080] Also, it is preferable that the enzyme of the present inventiontransfers GalNAc from a GalNAc donor to an oligosaccharide having GlcUAon its non-reduced terminal and also having a hyaluronic acid backbone,but does not substantially transfer GlcUA from a GlcUA donor.

[0081] (3) Influence by Metal Ions and the Like:

[0082] The enzyme of the present invention acts in the presence of Mn²⁺ion but does not substantially act in the presence of Ca²⁺ ion, Cu²⁺ ionor ethylenediaminetetraacetic acid.

[0083] Also, it is preferable that the enzyme of the present inventionfurther acts in the presence of Fe²⁺ or Mg²⁺ ion. Moreover, it ispreferable that the degree of action (enzyme activity) of the enzyme ofthe present invention in the presence of Mn²⁺ ion is higher than itsdegree of action (enzyme activity) in the presence of Fe²⁺ or Mg²⁺ ion.

[0084] Also, it was observed that when a reaction was carried out usingthe enzyme of the present invention at a temperature of 25° C. or more,size of the reaction product (chondroitin chain) became small as thereaction temperature increased (cf., Examples shown below). Accordingly,it is considered that enzyme activity of the enzyme of the presentinvention decreases as the reaction temperature increases at 25° C. ormore under the reaction conditions described in the following Examples.

[0085] It is preferable that the enzyme of the present invention isderived from Escherichia coli. Particularly, Escherichia coli strainhaving a gene related to the production of capsular polysaccharide ispreferable, and Escherichia coli strain whose capsular antigen (K) is“K4” is more preferable.

[0086] As the Escherichia coli strain whose capsular antigen serotype is“K4”, Escherichia coli strain K4 (Escherichia coli serotype 05:K4(L):H4)can be preferably exemplified, and more specifically, ATCC 23502, NCDCU1-41, Freiburg collection number 2616 and the like can be preferablyexemplified.

[0087] It is preferable also that the enzyme of the present invention isa protein selected from the following (A) and (B):

[0088] (A) a protein comprising the amino acid sequence represented bySEQ ID NO: 2;

[0089] (B) a protein comprising an amino acid sequence in which one or afew amino acid residue(s) in the amino acid sequence represented by SEQID NO: 2 are deleted, substituted, inserted or transposed, and having achondroitin polymerase activity.

[0090] Although mutation such as substitution, deletion, insertion,transposition or the like can occur in amino acid sequences of proteinsexisting in the nature caused by the modification reactions inside thecells or during purification of proteins after their formation, inaddition to polymorphism and mutation of DNA molecules encoding them, itis known that certain proteins having such mutations show physiologicaland biological activities which are substantially identical to thecorresponding proteins having no mutations. Such proteins which haveslight structural differences but no significant differences in theirfunctions are also included in the protein of the present invention. Acase in which the above mutation is artificially introduced into theamino acid sequence of protein is the same. In such a case, it ispossible to prepare larger varieties of mutants. For example, it isknown that a polypeptide prepared by substituting a cysteine residue inthe amino acid sequence of human interleukin 2 (IL-2) by a serineresidue maintains the interleukin 2 activity (Science, 224, 1431(1984)). Also, it is known that a certain protein has a peptide regionwhich is not essential for its activity. For example, a signal peptideexisting in a protein which is secreted into the extracellular moietyand a pro-sequence which can be found in a protease precursor or thelike correspond to this case, and most of these regions are removedafter translation of proteins or during their conversion into activeproteins. Such proteins are proteins which are present in differentforms in terms of their primary structure but finally have similarfunctions.

[0091] The term “a few amino acid residues” as used herein means thenumber of amino acids which may cause mutation in such a degree thatactivity of the chondroitin polymerase is not lost, and in the case of aprotein composed of 600 amino acid residues for example, it means thenumber of approximately from 2 to 30, preferably from 2 to 15, and morepreferably from 2 to 8.

[0092] Also, the protein of the present invention may contain an aminoacid sequence of other protein or peptide, so long as it contains theamino acid sequence of the above (A) or (B). That is, the protein of thepresent invention may be a fusion protein with other protein or peptide.

[0093] For example, fusion proteins of a protein comprising the aminoacid sequence described in the above (A) or (B) with a marker peptideand the like are also included in the protein of the present invention.Such proteins of the present invention have a merit in that theirpurification can be carried out easily. Examples of the above markerpeptide include protein A, insulin signal sequence, His, FLAG, CBP(calmodulin-binding protein), GST (glutathione S-transferase) and thelike. For example, its fusion protein with protein A can be purifiedconveniently by affinity chromatography using an IgG-linked solid phase.In the same manner, a solid phase to which magnetic nickel is linked canbe used for a fusion protein with His tag, and a solid phase to which ananti-FLAG antibody is linked can be used for a fusion protein with FLAG.Also, since a fusion protein with insulin signal is secreted into anextracellular moiety (a medium or the like), extraction operations suchas cell disintegration and the like become unnecessary. It is preferablethat the protein of the present invention (the enzyme of the presentinvention) is soluble.

[0094] Preferred examples include a fusion protein with a peptide (Histag) represented by the amino acid sequence represented by SEQ ID NO:11. It is preferable to carry out fusion of this His tag continuously ata position just before the amino acid sequence represented by SEQ ID NO:2. The fusion protein can be produced by expressing a DNA in which thenucleotide sequence represented by SEQ ID NO: 4 is connectedcontinuously to a position just before the nucleotide sequencerepresented by SEQ ID NO: 1. The fusion protein is soluble.

[0095] The “chondroitin polymerase activity” can be detected inaccordance with a generally used glycosyltransferase assay method.Specifically, it can be detected as the activity to synthesizechondroitin by transferring GlcUA and GalNAc alternately to anon-reduced terminal of a sugar chain (acceptor), using a GlcUA donor, aGalNAc donor and a sugar chain which becomes the acceptor.

[0096] For example, when GlcUA is transferred from a GlcUA donor to asugar chain having GalNAc on its non-reduced terminal, and GalNAc istransferred from a GalNAc donor to a sugar chain having GlcUA on itsnon-reduced terminal, it can be judged that it has an activity totransfer GlcUA and GalNAc alternately to a non-reduced terminal of thesugar chain, namely the chondroitin polymerase activity. It ispreferable to employ the enzyme activity measuring method described inthe following Examples. By such a method, deletion, substitution,insertion or transposition of amino acids keeping the chondroitinpolymerase activity can be selected easily.

[0097] Production processes of the enzyme of the present invention andprotein of the present invention are not particularly limited, and theycan be produced by expressing the DNA of the present invention describedbelow in appropriate cells. Also, those which are isolated from naturalresources and produced by chemical synthesis and the like are includedin the enzyme of the present invention and the protein of the presentinvention as a matter of course. The production processes of the enzymeof the present invention (the protein of the present invention) usingthe DNA of the present invention will be described later.

[0098] <2> DNA of the Present Invention

[0099] The DNA of the present invention is a DNA comprising any one ofthe following (a) to (c):

[0100] (a) a DNA encoding a protein consisting of the amino acidsequence represented by SEQ ID NO: 2;

[0101] (b) a DNA encoding a protein consisting of an amino acid sequencein which one or a few amino acid residue(s) in the amino acid sequencerepresented by SEQ ID NO: 2 are deleted, substituted, inserted ortransposed, and having a chondroitin polymerase activity;

[0102] (c) a DNA which hybridizes with the DNA in (a), a DNAcomplementary to the DNA in (a) or a DNA having a part of nucleotidesequences of the DNA in (i) and (ii) under stringent conditions.

[0103] The DNA is preferably represented by SEQ ID NO: 1.

[0104] The “stringent conditions” as used herein mean conditions underwhich a so-called specific hybrid is formed but a non-specific hybrid isnot formed (cf. Sambrook, J. et al., Molecular Cloning, A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press (1989) andthe like). Examples of the “stringent conditions” include conditions inwhich the hybridization is carried out at 42° C. in a solutioncontaining 50% formamide, 4×SSC, 50 mM HEPES (pH 7.0), 10×Denhardt'ssolution and 100 μg/ml sermon sperm DNA, and the product is washed with2×SSC, 0.1% SDS solution at room temperature and then with 0.1×SSC, 0.1%SDS solution at 50° C.

[0105] The DNA of the present invention is originally obtained fromEscherichia coli strain having K4 antigen, but DNA samples obtained fromother transformed organism species and produced by chemical synthesisand the like are also included therein as a matter of course. Productionprocesses of the DNA of the present invention are not particularlylimited too, but it is preferable to use, e.g., the process described inthe following Examples.

[0106] It is easily understood by those skilled in the art that DNAshaving various different nucleotide sequences due to degeneracy ofgenetic code are present as the DNA of the present invention.

[0107] <3> Vector of the Present Invention

[0108] The vector of the present invention is a vector comprising theDNA of the present invention. Preferable DNA of the present invention inthe vector of the present invention is the same as described in theabove <2>. Also, since the vector of the present invention is preferablyused in the enzyme production process of the present invention whichwill be described later, it is preferably an expression vector.

[0109] The vector of the present invention can be prepared by insertingthe DNA of the present invention into a known vector.

[0110] As the vector into which the DNA of the present invention isinserted, for example, an appropriate vector which can express theintroduced DNA (a phage vector, plasmid vector or the like) can be used,and it can be optionally selected in response to each host cell intowhich the vector of the present invention is inserted. Examples of thehost-vector system include a combination of a mammal cell such as COScell, 3LL-HK46 or the like with an expression vector for mammal cellsuch as pGIR201 (Kitagawa, H. and Paulson, J. C., J. Biol. Chem., 269,1394-1401 (1994)), pEF-BOS (Mizushima, S. and Nagata, S. Nucleic AcidRes., 18, 5322 (1990)), pCXN2 (Niwa, H., Yamamura, K. and Miyazaki, J.Gene, 108, 193-200 (1991)), pCMV-2 (manufactured by Eastman Kodak),pCEV18, pME18S (Maruyama et al., Med. Immunol., 20, 27 (1990)), pSVL(manufactured by Pharmacia Biotech) or the like and a combination ofEscherichia coli with a expression vector for procaryotic cell such aspTrcHis (manufactured by Invitrogen), pGEX (manufactured by PharmaciaBiotech), pTrc99 (manufactured by Pharmacia Biotech), pKK233-3(manufactured by Pharmacia Biotech), pEZZZ18 (manufactured by PharmaciaBiotech), pCH110 (manufactured by Pharmacia Biotech), pET (manufacturedby Stratagene), pBAD (manufactured by Invitrogen), pRSET (manufacturedby Invitrogen), pSE420 (manufactured by Invitrogen) or the like.Additionally, an insect cell, yeast, Bacillus subtilis and the like canalso be exemplified as the host cell and various vectors correspondingthereto can be exemplified. Among the above host-vector systems, acombination of Escherichia coli with pTrcHis is preferable.

[0111] Also, as the vector into which the DNA of the present inventionis inserted, a vector constructed in such a manner that it expresses afusion protein of the protein of the present invention (enzyme of thepresent invention) with a marker peptide can also be used, which, asdescribed in the above <1>, is particularly preferable when thechondroitin polymerase expressed using the vector of the presentinvention is purified. Specifically, a vector comprising aHis-expressing nucleotide sequence (e.g., the nucleotide sequencerepresented by SEQ ID NO: 4) is preferable.

[0112] When any of the above vectors is used, the DNA of the presentinvention can be connected with the vector after treating both of themwith restriction enzymes and the like and optionally carrying outsmooth-ending and connection of a cohesive end so that connection of theDNA of the present invention with the vector becomes possible.

[0113] As the process for producing the vector of the present invention,for example, the process described in the following Examples can be usedand is preferable.

[0114] <4> Transformant of the Present Invention

[0115] The transformant of the present invention is a transformant inwhich a host is transformed with the vector of the present invention.

[0116] The “host” as used herein may be any host in which recombinationby the vector of the present invention can be carried out but ispreferably one which can exert function of the DNA of the presentinvention or a recombinant vector into which the DNA of the presentinvention is inserted. Examples of the host include animal cells, plantcells and microbial cells are included, and mammal cells such as COScells (COS-1 cell, COS-7 cells and the like), 3LL-HK46 cell, etc.,Escherichia coli, insect cells, yeast, Bacillus subtilis and the like.The host can be optionally selected in response to each vector of thepresent invention, but, for example, when a vector of the presentinvention prepared based on pTrcHis is used, it is preferable to selectEscherichia coli strain.

[0117] The host can be transformed by the vector of the presentinvention in the usual way. For example, the host can be transformed byintroducing the vector of the present invention into the host by amethod using a commercially available transfection reagent, aDEAE-dextran method, electroporation or the like.

[0118] The transformant of the present invention obtained in this mannercan be used in the enzyme production process of the present inventiondescribed below.

[0119] <5> Enzyme Production Process of the Present Invention

[0120] The enzyme production process of the present invention is aprocess for producing a chondroitin polymerase, comprising growing thetransformant of the present invention; and collecting a chondroitinpolymerase from the grown material.

[0121] The term “growing” as used herein means a general idea whichincludes growth of cells or microorganism as the transformant of thepresent invention itself and growth of an animal, insect or the likeinto which cells as the transformant of the present invention areincorporated. Also, the term “grown material” as used herein means aconcept which includes a medium (supernatant of culture medium) andcultured host cells after growth of the transformant of the presentinvention, secreted matter, excreted matter and the like.

[0122] Growth conditions (medium, culture condition and the like) areappropriately selected based on the host to be used.

[0123] According to the enzyme production process of the presentinvention, various forms of chondroitin polymerase can be produced basedon the transformant to be used.

[0124] For example, when a transformant transformed with an expressionvector constructed for expressing a fusion protein with a marker peptideis grown, a chondroitin polymerase fused with the marker peptide isproduced. Specifically, for example, a chondroitin polymerase fused withHis tag is produced by growing a transformant transformed with anexpression vector constructed for effecting expression of a protein inwhich the amino acid sequence represented by SEQ ID NO: 11 iscontinuously fused to a position just before the amino acid sequencerepresented by SEQ ID NO: 2. Particularly, it is preferable to use atransformant transformed with an expression vector constructed byconnecting the nucleotide sequence represented by SEQ ID NO: 4continuously to a position just before the nucleotide sequencerepresented by SEQ ID NO: 1.

[0125] The chondroitin polymerase can be collected from the grown matterby known protein extraction and purification methods based on the formof the produced chondroitin polymerase.

[0126] For example, when the chondroitin polymerase is produced in asoluble form secreted into a medium (supernatant of culture medium), themedium may be collected and used directly as the chondroitin polymerase.Also, when the chondroitin polymerase is produced in a soluble formsecreted into the cytoplasm or in an insoluble form (membrane binding),the chondroitin polymerase can be extracted by cell disintegration suchas a method using a nitrogen cavitation apparatus, homogenization, glassbeads mill, sonication, an osmotic shock method, freezing-thawing, etc.,surfactant extraction, a combination thereof or the like, and theextract may be used directly as the chondroitin polymerase.

[0127] The chondroitin polymerase can be further purified from the mediaor extracts, which is preferable. The purification may be eitherimperfect purification (partial purification) or perfect purification,which can be appropriately selected based on, e.g., the object using thechondroitin polymerase, and the like.

[0128] Examples of the purification method include salting out byammonium sulfate, sodium sulfate, etc., centrifugation, dialysis,ultrafiltration, adsorption chromatography, ion exchange chromatography,hydrophobic chromatography, reverse phase chromatography, gelfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, a combination thereof and the like.

[0129] Production of the chondroitin polymerase can be confirmed byanalyzing its amino acid sequence, actions, substrate specificity andthe like.

[0130] <6> Synthesizing Agent of the Present Invention

[0131] The synthesizing agent of the present invention is a sugar chainsynthesizing agent, comprising an enzyme protein which comprises anamino acid sequence represented by the following (A) or (B) and hasenzymic activities of the following (i) and (ii):

[0132] (A) the amino acid sequence represented by SEQ ID NO: 2;

[0133] (B) an amino acid sequence in which one or a few two amino acidresidue(s) in the amino acid sequence represented by SEQ ID NO: 2 aredeleted, substituted, inserted or transposed;

[0134] (i) GlcUA and GalNAc are alternately transferred to a non-reducedterminal of a sugar chain from a GlcUA donor and a GalNAc donor,respectively;

[0135] (ii) GlcNAc is transferred to a non-reduced terminal of a sugarchain having GluUA on the non-reduced terminal from a GlcNAc donor.

[0136] As the “enzyme protein which comprises an amino acid sequencerepresented by the following (A) or (B) and has enzymic activities ofthe following (i) and (ii)” which is the active ingredient of thesynthesizing agent of the present invention, the enzyme of the presentinvention or the protein of the present invention can be used as such.

[0137] The synthesizing agent of the present invention is a result ofapplying the “GalNAc transferring action”, “GlcNAc transferring action”and “GlcUA transferring action” of the enzyme of the present inventionand protein of the present invention as a sugar chain synthesizingagent.

[0138] The synthesizing agent of the present invention is used for thesynthesis of sugar chains. The term “synthesis of sugar chain” or “sugarchain synthesis” as used herein means a concept including elongation ofa certain sugar chain by transferring and adding a monosaccharide to thesugar chain. For example, a concept of transferring and adding amonosaccharide such as GlcUA, GalNAc, GlcNAc or the like to a sugarchain such as chondroitin, chondroitin sulfate, hyaluronic acid and thelike to elongate the sugar chain is included in the term “sugar chainsynthesis” as used herein.

[0139] The form of the synthesizing agent of the present invention isnot limited, and it may be any one of a solution form, a frozen form, afreeze-dried form and an immobilized enzyme form in which it is linkedto a carrier. Also, it may contain other components (e.g.,pharmaceutically acceptable carrier, carrier which is acceptable forreagent, etc.), so long as they do not have influence on the activity ofchondroitin polymerase.

[0140] <7> Sugar Chain Production Process of the Present Invention

[0141] The sugar chain production process of the present invention usesthe synthesizing agent of the present invention and is divided into thefollowing five methods in response to the sugar donors and acceptorsubstrates.

[0142] (1) Sugar Chain Production Process of the Present Invention 1

[0143] A process for producing a sugar chain represented by thefollowing formula (3), which comprises at least a step of allowing thesynthesizing agent of the present invention to contact with a GalNAcdonor and a sugar chain represented by the following formula (1):

GlcUA-X—R¹   (1)

GalNAc-GlcUA-X—R¹   (3)

[0144] (2) Sugar Chain Production Process of the Present Invention 2

[0145] A process for producing a sugar chain represented by thefollowing formula (4), which comprises at least a step of allowing thesynthesizing agent of the present invention to contact with a GlcNAcdonor and a sugar chain represented by the following formula (1):

GlcUA-X—R¹   (1)

GlcNAc-GlcUA-X—R¹   (4)

[0146] (3) Sugar Shain Production Process of the Present Invention 3

[0147] A process for producing a sugar chain represented by thefollowing formula (5), which comprises at least a step of allowing thesynthesizing agent of the present invention to contact with a GlcUAdonor and a sugar chain represented by the following formula (2):

GalNAc-GlcUA-R²   (2)

GlcUA-GalNAc-GlcUA-R²   (5)

[0148] (4) Sugar Chain Production Process of the Present Invention 4

[0149] A process for producing a sugar chain selected from the followingformulae (6) and (8), which comprises at least a step of allowing thesynthesizing agent of the present invention to contact with a GalNAcdonor and a GlcUA donor and a sugar chain represented by the followingformula (1):

GlcUA-X—R¹   (1)

(GlcUA-GalNAc)n-GlcUA-X—R¹   (6)

GalNAc-(GlcUA-GalNAc)n-GlcUA-X—R¹   (8)

[0150] (5) Sugar Chain Production Process of the Present Invention 5

[0151] A process for producing a sugar chain selected from the followingformulae (7) and (9), which comprises at least a step of allowing thesynthesizing agent of the present invention to contact with a GalNAcdonor, a GlcUA donor and a sugar chain represented by the followingformula (2):

GalNAc-GlcUA-R²   (2)

(GalNAc-GlcUA)n-GalNAc-GlcUA-R²   (7)

GlcUA-(GalNAc-GlcUA)n-GalNAc-GlcUA-R²   (9)

[0152] In each of the formulae, X represents GalNAc or GlcNAc, and R¹and R² each represents any group. R¹ and R² are the same or differentfrom each other.

[0153] Examples of R¹ and R² include a sugar chain having a chondroitinbackbone, a sugar chain having a hyaluronic acid backbone and the like.

[0154] The sugar chain represented by formula (1) is preferablychondroitin sulfate (particularly chondroitin sulfate C), chondroitin orhyaluronic acid having GlcUA on its non-reduced terminal, or anoligosaccharide thereof.

[0155] The sugar chain represented by formula (2) is preferablychondroitin sulfate (particularly chondroitin sulfate C) or chondroitinhaving GalNAc on its non-reduced terminal, or an oligosaccharidethereof.

[0156] As the GalNAc donor, nucleoside diphosphate-GalNAc is preferable,and UDP-GalNAc is particularly preferable. Furthermore, as the GlcNAcdonor, nucleoside diphosphate-GlcNAc is preferable, and UDP-GlcNAc isparticularly preferable. Moreover, as the GlcUA donor, nucleosidediphosphate-GlcUA is preferable, and UDP-GlcUA is particularlypreferable.

[0157] The method for carrying out “contact” is not particularlylimited, so long as the enzyme reaction is generated by mutual contactof molecules of the enzyme of the present invention (or the protein ofthe present invention) contained in the synthesizing agent of thepresent invention, a donor and an acceptor (sugar chain). For example,the contact may be carried out in a solution in which the threecomponents are dissolved. Also, the enzyme reaction can be carried outcontinuously using an immobilized enzyme in which chondroitin polymerasecontained in the synthesizing agent of the present invention is linkedto an appropriate solid phase (beads or the like) or a membrane typereactor using ultrafiltration membrane, dialysis membrane or the like.Also, the enzyme reaction can be carried out by linking the acceptor toa solid phase similar to the method described in WO 00/27437. Inaddition, a bioreactor which regenerate (synthesize) the donor may beused in combination.

[0158] In addition, in the above processes (4) and (5), it is not alwaysnecessary to contact the GalNAc donor and GlcUA donor simultaneouslywith the synthesizing agent of the present invention and the sugar chainrepresented by formula (1) or (2), and the donors may be allowed tocontact alternately.

[0159] The conditions for carrying out the enzyme reaction is notparticularly limited, so long as they are conditions under which theenzyme of the present invention (or the protein of the presentinvention) can function, but it is preferable to carry out the reactionat around neutral pH (e.g., about pH 7.0 to 7.5), and it is morepreferable to carry out the reaction in a buffer having the bufferingaction under the pH. Also, the temperature in this case is notparticularly limited, so long as the activity of the enzyme of thepresent invention (or the protein of the present invention) is retained,but a temperature of approximately from 25° C. to 30° C. can beexemplified. Also, when there is a substance which increases theactivity of the enzyme of the present invention (or the protein of thepresent invention), the substance may be added. For example, it ispreferable to allow Mn²⁺ and the like to coexist. The reaction time canbe determined optionally by those skilled in the art in response to theamounts of the synthesizing agent of the present invention, donor andacceptor to be used and other reaction conditions.

[0160] Isolation and the like of sugar chain from the formed product canbe carried out by known methods.

[0161] Also, a sulfated saccharide such as chondroitin sulfate or thelike can be produced by using the synthesizing agent of the presentinvention (chondroitin polymerase) and a sulfotransferase incombination.

[0162] For example, a sulfated saccharide such as chondroitin sulfate orthe like can be produced by simultaneously carrying out formation ofchondroitin and transfer of sulfate group in the above sugar chainproduction process, by further allowing a sulfate group donor(3′-phosphoadenosine 5′-phosphosulfate (PAPS) or the like) and asulfotransferase to coexist. The sulfotransferase may be used as animmobilized enzyme by linking it to an appropriate solid phase (beads orthe like) similar to the above case or allowed to react continuouslyusing a membrane type reactor using ultrafiltration membrane, dialysismembrane or the like. In this case, a bioreactor which regenerate(synthesize) the sulfate group donor may be used in combination.

[0163] The sulfotransferase which can be used herein may be any enzymewhich can transfer a sulfate group to chondroitin and can beappropriately selected from known enzymes based on the kind of desiredchondroitin sulfate. Also, two or more kinds of sulfotransferase havingdifferent sulfate group transferring positions may be used incombination.

[0164] Chondroitin 6-O-sulfotransferase (J. Biol. Chem., 275(28),21075-21080 (2000)) can be exemplified as the sulfotransferase. However,there is no limitation thereto and other enzymes can also be used.

[0165] <8> Probe of the Present Invention

[0166] The probe of the present invention is a hybridization probecomprising a nucleotide sequence complementary to the nucleotidesequence represented by SEQ ID NO: 1 or a part thereof.

[0167] The probe of the present invention can be obtained by preparingan oligonucleotide comprising a nucleotide sequence complementary to thenucleotide sequence represented by SEQ ID NO: 1 or a part thereof, andlabeling it with a label suitable for hybridization (e.g.,radioisotope).

[0168] The size of the oligonucleotide is appropriately selected basedon conditions and the like of the hybridization using the probe of thepresent invention.

[0169] It is expected that the probe of the present invention becomes auseful tool for examining biological functions of chondroitin sulfate.This is because chondroitin sulfate is broadly expressed and plays animportant role in a large number of tissues, particularly in the brain.It is considered that the probe is also useful in searching for therelationship between genes and diseases.

[0170] <9> Catalyst of the Present Invention

[0171] The catalyst of the present invention is a glycosyltransfercatalyst which comprises an enzyme protein comprising an amino acidsequence selected from the following (A) and (B), and is capable oftransferring GlcUA, GalNAc and GlcNAc from a GlcUA donor, a GalNAc donorand a GlcNAc donor, respectively to a non-reduced terminal of a sugarchain:

[0172] (A) the amino acid sequence represented by SEQ ID NO: 2;

[0173] (B) an amino acid sequence in which one or a few amino acidresidue(s) in the amino acid sequence represented by SEQ ID NO: 2 aredeleted, substituted, inserted or transposed.

[0174] As the “enzyme protein comprising an amino acid sequencerepresented by (A) or (B)” which is the active ingredient of thecatalyst of the present invention, the enzyme of the present inventionor protein of the present invention can be used as such.

[0175] The catalyst of the present invention is a result of applying the“GalNAc transferring action”, “GlcNAc transferring action” and “GlcUAtransferring action” of the enzyme of the present invention and proteinof the present invention as a glycosyltransfer catalyst.

[0176] The catalyst of the present invention can be used for transfer ofGlcUA, GalNAc or GlcNAc. For example, it can be used for transferring amonosaccharide such as GlcUA, GalNAc, GlcNAc or the like to anon-reduced terminal of a sugar chain such as chondroitin, chondroitinsulfate, hyaluronic acid or the like.

[0177] The form of the catalyst of the present invention is not limited,and it may be any one of a solution form, a frozen form, a freeze-driedform and an immobilized enzyme form in which it is linked to a carrier.Also, it may contain other components (e.g., pharmaceutically acceptablecarrier, carrier which is acceptable for reagent, etc.), so long as theydo not have influence on the transferring activity of GlcUA, GalNAc orGlcNAc.

[0178] Since the enzyme of the present invention and the protein of thepresent invention can transfer GlcUA and GalNAc alternately as a singlemolecule, they are markedly useful as tools for the large scaleproduction of sugar chain having a chondroitin backbone (chondroitin,chondroitin sulfate and the like), as the active ingredients of thesynthesizing agent of the present invention and the catalyst of thepresent invention, reagents or the like. Also, the DNA of the presentinvention is markedly useful as a tool for the large scale production ofsuch enzyme of the present invention and protein of the presentinvention. The vector of the present invention is markedly useful,because it can retain the DNA of the present invention stably andfunction effectively and efficiently. The transformant of the presentinvention is also markedly useful, because not only it can retain thevector of the present invention stably and function effectively andefficiently, but also it can be used as such for the large scaleproduction of the enzyme of the present invention and the protein of thepresent invention. In addition, the enzyme production process of thepresent invention is markedly useful for the large scale production ofthe enzyme of the present invention and protein of the presentinvention. Also, the sugar chain production process of the presentinvention is markedly useful for the large scale production of sugarchain having the chondroitin backbone (chondroitin, chondroitin sulfateand the like). The synthesizing agent of the present invention and thecatalyst of the present invention are markedly useful, because they canbe used in the sugar chain production process of the present invention.

[0179] Since high quality and uniform chondroitin polymerase can beproduced conveniently, quickly and in a large scale by the presentinvention, low cost products can be provided for the industrial fieldand therefore the present invention has markedly high availability.

[0180] The present invention is explained in detail based on Examples.However, the present invention is not limited thereto.

[0181] UDP-[¹⁴C]GlcUA, UDP-[³H]GalNAc and UDP-[¹⁴C]GlcNAc used inExamples were purchased from NEN Life Sciences. Also, UDP-GlcUA,UDP-GalNAc and UDP-GlcNAc were purchased from Sigma.

EXAMPLE 1

[0182] Cloning of Chondroitin Polymerase Gene:

[0183] (1) Preparation of DNA Library

[0184]Escherichia coli strain K4 (serotype 05:K4(L):H4, ATCC 23502) wascultured at 37° C. overnight in 50 ml of LB medium. The cells werecollected by centrifugation (3,800 rpm, 15 minutes), suspended in 9 mlof 10 mM Tris-HCl (pH 8.0) buffer containing 1 mMethylenediaminetetraacetic acid (EDTA) (hereinafter referred to as “TE”)and then treated at 37° C. for 1 hour by adding 0.5 ml of 10% SDS and 50μl of proteinase K (20 mg/ml, Boehringer Mannheim). To the suspension,10 ml of PCI solution (phenol:chloroform:isoamyl alcohol=25:24:1) wasadded, followed by stirring for 30 minutes, and the resulting mixturewas centrifuged (3,800 rpm, 15 minutes) to collect the water layer andthe intermediate layer insoluble matter and again centrifuged (10,000rpm, 30 minutes). The supernatant was recovered and 50 μl of RNase A (20mg/ml, Sigma) was added thereto for reaction at 37° C. for 1 hour. Tothe treated solution, 10 ml of PCI solution was added, followed bystirring for 30 minutes, and the resulting mixture was centrifuged(3,800 rpm, 15 minutes) to collect the water layer and again centrifuged(10,000 rpm, 30 minutes). The supernatant was recovered and dialyzedagainst 2,000 ml of TE at 4° C. overnight, and the thus dialyzedsolution (7.5 ml) was used as a chromosomal DNA solution (DNAconcentration, 0.9 mg/ml). The thus obtained K4 strain-derivedchromosomal DNA solution (120 μl) was digested using a restrictionenzyme Sau3A1 (4 units: NEB) at 37° C. for 30 minutes and then subjectedto 0.3% agarose gel electrophoresis, and then the agarose gelcorresponding to the DNA of about 7 to 11 kbp was cut out. The gel thuscut out was put into a 1.5 ml capacity tube having a hole on its bottompricked with a needle and, together with the tube, inserted into a 2 mlcapacity tube and centrifuged (8,000 rpm, 1 minute) to break up the gel.Neutralized phenol in an almost the same volume of the gel was addedthereto, followed by vigorously stirring and then the resulting mixturewas frozen at −80° C. Thirty minutes thereafter, the temperature wasreturned to room temperature to melt the mixture, followed bycentrifugation (13,000 rpm, 5 minutes). The resulting aqueous layer wascollected, the same volume of PCI solution was added thereto, followedby stirring, and then the resulting mixture was centrifuged (13,000 rpm,5 minutes). The aqueous layer was collected, {fraction (1/10)} volume of3 M sodium acetate solution and the same volume of 2-propanol were addedthereto to precipitate DNA, and the precipitate was then collected bycentrifugation (13,000 rpm, 30 minutes). To the thus collectedprecipitate, 70% ethanol solution was added, followed by centrifugation(13,000 rpm, 5 minutes), and then the resulting precipitate wasdissolved by adding 100 μl of TE. In order to concentrate the resultingsolution, DNA was precipitated by adding 10 μl of 3 M sodium acetatesolution and 300 μl of ethanol and then recovered by centrifugation(13,000 rpm, 20 minutes). To the thus collected precipitate, 70% ethanolsolution was added, followed by centrifugation (13,000 rpm, 5 minutes),and the resulting precipitate was dissolved in 4 μl of purified water toobtain a chromosomal DNA fragment solution. The DNA fragment solution (2μl) was inserted into a λ phage vector (λ EMBL3: STRATAGENE) which hadbeen treated with restriction enzymes (BamHI (80 units: NEB) and EcoRI(80 units: NEB)) and subjected to packaging using a packaging kit(Gigapack III Gold Packaging Extract, STRATAGENE) in accordance with themanufacture's instructions, and then Escherichia coli strain (NM539) wasinfected with the λ phage and propagated to prepare a K4 chromosomal DNAlibrary.

[0185] (2) Preparation of Probe

[0186] Among 3 regions of the K antigen gene cluster moiety ofEscherichia coli strain K5 (serotype O10:K5(L):H4, ATCC 23506) havingknown sequences (Mol. Microbiol., 17(4), 611-620 (1995)), whileinterposing the K antigen polysaccharide-specific region R-II (gene bankaccession NO. X77617), a primer set (CS-S5′-ACCCAACACTGCTACAACCTATATCGG-3′ (SEQ ID NO: 5); CS-AS5′-GCGTCTTCACCAATAAATACCCACGAAACT-3′ (SEQ ID NO: 6)) to obtain a DNAfragment of about 1 kbp from the 3′-terminal of the R-I region (genebank accession NO. X74567), and another primer set (TM-S5′-CGAGAAATACGAACACGCTTTGGTAA-3′ (SEQ ID NO: 7); TM-AS5′-ACTCAATTTTCTCTTTCAGCTCTTCTTG-3′ (SEQ ID NO: 8)) to obtain a DNAfragment of about 1 kbp from the 5′-terminal of the R-III region (genebank accession NO. X53819) were selected and prepared.

[0187] Using the respective primer sets for R-I and R-III and using, asthe template, genome DNA fragments of the strain K4 extracted andpurified after Sau3A1 treatment and subsequent agarose gelelectrophoresis in the above (1), polymerase chain reaction (PCR) (94°C., 1 min-(94° C., 45 sec- 47° C., 30 sec-72° C., 5 min) 30 cycles-72°C., 10 min (for R-I), 94° C., 1 min-(94° C., 45 sec-50° C., 30 sec-72°C., 5 min) 30 cycles-72° C., 10 min (for R-III)) was carried out toobtain K4-derived R-I region 1.3 kbp (K4RI3) and R-III region 1.0 kbp(K4RIII5) DNA fragments. Nucleotide sequences of the thus obtained DNAfragments were determined using ABI PRISM 310 Genetic Analyzer(Perkin-Elmer). The homology with the strain K5 DNA at the same geneticpositions was 96% and 95%, respectively.

[0188] (3) Gene Cloning of K4R-II Region

[0189] Using respective R-I region and R-III region DNA fragments (K4RI3and K4RIII5) as probes, K4 antigen gene clusters were screened from theK4 chromosomal DNA library obtained in the above (1). Escherichia coli(strain NM539) culture (30 μl) was infected with the K4 chromosomal DNAlibrary (λ phage 40 μl) (37° C., 15 minutes), 10 ml of top agarose wasadded thereto, the resulting mixture was spread on LB plate mediumcontained in five 10×14 cm Petri dishes, followed by culturing at 37° C.for 9.5 hours to form plaques. Two 9×13 cm membranes (Hybond-N+:Amersham Pharmacia Biotech) were prepared for each plate, and the firstand second membranes were put on the medium for 1 minute and 3 minutes,respectively. After removing excess moisture, each membrane was soakedfor 2 minutes in 0.5 M NaOH solution containing 1.5 M NaCl to carry outdenaturation treatment and then soaked for 3 minutes in 1 M Tris-HCl (pH7.4) containing 1.5 M NaCl to carry out neutralization treatment. Afterdrying, the membrane was baked at 80° C. for 2 hours to prepare afilter. The filter was subjected to pre-hybridization treatment at 65°C. for 1 hour, hybridized with [³²P]-labeled K4RI3 at 64° C. overnight(15 hours) in 0.5 M Church phosphate buffer (pH 7.2), 1 mM EDTA and 7%SDS, and then washed three times with 40 mM Church phosphate buffer (pH7.2) containing 1% SDS (65° C., each for 15 minutes). The filter wasdried and then exposed to an X-ray film to thereby obtain 30 positiveplaques. The presence of K4RI3 was confirmed for each of them by PCR,and 7 plaques among them were subjected to the second screening. Next,the filter hybridized with K4RI3 was boiled in 0.5% SDS solution for 3minutes to remove K4RI3 and then dried to be used as a K4RIII5hybridization filter. The filter was subjected to pre-hybridizationtreatment at 65° C. for 1 hour, hybridized with [³²P]-labeled K4RIII5 at64° C. overnight and then washed three times with 40 mM Church phosphatebuffer containing 1% SDS. The filter was dried and then exposed to anX-ray film to thereby obtain 29 positive plaques. The presence ofK4RIII5 was confirmed for each of them by PCR, and 18 plaques among themwere subjected to the second screening. In the second screening, LBplate medium of φ 9 cm was used, and positive plaques were obtained bythe same method of first screening.

[0190] After the first and second screening, 4 λ phage clones wereobtained from the R-I region, and 10 clones from the R-III region. Eachof the clones was subjected to enzyme treatment with EcoRI (10 units:NEB), SalI (10 units: NEB) and BamHI (10 units: NEB), each independentlyor simultaneously in various combinations, and their restriction mapswere prepared based on the size of fragments observed by electrophoresis(FIG. 1).

[0191] Among these clones, one clone (CS23, insertion region 15.4 kbp)is a DNA clone prepared based on the R-III region probe, but since italso showed a weak reaction with the R-I region probe, 5′-terminalsequence of the insertion region was examined to find a sequencecompletely coincided with the 3′-end of the R-I region probe. Since bothof the DNA fragments of the R-I region and R-III region were containedin the insertion region, the clone was judged as a clone which containsall of the R-II region of the K antigen gene cluster of the strain K4.

[0192] (4) Genetic Analysis of K4 R-II Region

[0193] Subcloning of the above CS23 clone was carried out to carry outits sequencing. First, each of about 3 kbp and 8 kbp DNA fragmentsobtained by treating the CS23 clone with EcoRI and 2 kbp, 5 kbp and 7kbp DNA fragments obtained by treating it with SalI was ligated with acloning vector (pBluescript II KS(−)) and integrated into Escherichiacoli strain (XLI-Blue) to obtain a clone having different direction ofinsert. By repeating “treatment of multi-cloning sites of the vectorwith various restriction enzymes-ligation-transformation” on the clone,22 plasmids having partial DNA fragments of the R-II region wereobtained. By carrying out sequencing of the insertion DNA fragments andconnecting them, complete gene sequence of the K4 R-II region wasdetermined (SEQ ID NO: 3).

[0194] (5) Identification of Chondroitin Polymerase Gene

[0195] As a result of the analysis of the K4 strain R-II region DNAsequence, the presence of 8 open reading frames (ORF) was predicted(FIG. 2).

[0196] Among them, the third position ORF counting from the R-III side(2,061 bp (nucleotide numbers 3,787 to 5,847 in SEQ ID NO: 3, sequenceof 2,058 bp by excluding the termination codon is shown in SEQ ID NO:1), 686 as the number of amino acids, molecular weight obtained bycalculation 79,256 (SEQ ID NO: 2)) showed 59% of homology with aPasteurella multocida hyaluronic acid synthase (class 2 type pmHAS; J.Biol. Chem., 273(14), 8454-8458 (1998)). Also, the first position ORFcounting from the R-III side (1,017 bp (nucleotide numbers 643 to 1,659in SEQ ID NO: 3), 339 as the number of amino acids) showed 60% ofhomology with Pasteurella multocida UDP-glucose-4-epimerase (Submitted(Oct. 29, 1996) Genetics and Microbiology, Autonomus University ofBarcelona, Edifici C, Bellaterra, BCN 08193, Spain), the fourth positionORF (1,332 bp (nucleotide numbers 5,877 to 7,207 in SEQ ID NO: 3))showed high homology (98%) with Insertion Sequence 2 (Nucleic AcidsRes., 6(3), 1111-1122 (1979)), and the seventh position ORF (1,167 bp(nucleotide numbers 11,453 to 12,619 in SEQ ID NO: 3), 389 as the numberof amino acids) showed 65% of homology with the kfiD (Mol. Microbiol.,17(4), 611-620 (1995)) gene (encodes UDP-glucose dehydrogenase) ofEscherichia coli strain (K5). Also, since the eighth position ORF (1,035bp (nucleotide numbers 13,054 to 14,088 in SEQ ID NO: 3), 345 as thenumber of amino acids) contained a DXD motif common toglycosyltransferase, it was considered that it has a sugar transferringactivity. Regarding the remaining three ORFs (Nos. 2, 5 and 6(nucleotide numbers 1,849 to 3,486, 7,210 to 8,673 and 9,066 to 10,631,respectively, in SEQ ID NO: 3), no homology was found.

EXAMPLE 2

[0197] Expression and Enzyme Activity of Chondroitin Polymerase Protein:

[0198] (1) In order to confirm that the K4 R-II region ORF (No. 3) is achondroitin polymerase gene, primers having restriction enzyme cut sitesand interposing the corresponding ORF moiety (K4C-SP5′-CGGGATCCCGATGAGTATTCTTAATCAAGC-3′ (SEQ ID NO: 9); K4C-AS5′-GGAATTCCGGCCAGTCTACATGTTTATCAC-3′ (SEQ ID NO: 10)) were prepared andPCR (94° C., 1 min-(94° C., 30 sec-59° C., 30 sec-74° C., 3 min) 20cycles-74° C., 10 min) was carried out. The PCR product was subjected to0.7% agarose gel electrophoresis and extracted and purified using a gelextraction kit (QIAGEN). After treating with restriction enzymes (BamHIand EcoRI), the product was again subjected to 0.7% agarose gelelectrophoresis and extracted and purified in the same manner to be usedas an insert.

[0199] The insert prepared in the above was inserted into an expressionvector (pTrcHisC: Invitrogen; containing the nucleotide sequencerepresented by SEQ ID NO: 4) which had been treated with restrictionenzymes (BamHI and EcoRI) and CIP, at 16° C. spending 1 hour in thepresence of T4 DNA ligase, and transformed into Escherichia coli strain(TOP10). By culturing the Escherichia coli strain (LB plate mediumcontaining ampicillin, 37° C., overnight), 7 colonies were obtained. Oneclone containing a plasmid into which the above insert was correctlyinserted was selected from them. The Escherichia coli was cultured (37°C., overnight) in 1.5 ml of LB medium containing ampicillin (100 μg/ml),and 50 μl of the cultured cell suspension was inoculated into 50 ml ofLB medium containing ampicillin (100 μg/ml) and cultured at 37° C. untilOD₆₀₀ became 0.6. To the culture, 1 ml of 0.5 M isopropyl1-thio-β-D-galactoside (IPTG) was added (final concentration: 1 mM) andinduction was carried out at 37° C. for 3 hours. The cells werecollected by centrifugation (10,000 rpm, 30 minutes) and suspended byadding 4 ml of a lysis buffer (50 mM NaH₂PO₄ (pH 8.0) containing 300 mMNaCl and 10 mM imidazole). To the suspension, 4 mg of lysozyme (Sigma)was added, the resulting mixture was allowed to stand on ice for 30minutes, and then the cells were disrupted by three times ofultrasonication, each for 10 seconds, using a sonicator. The supernatantwas collected by centrifugation (10,000 rpm, 30 minutes) and applied toNi-NTA agarose column (carrier 1 ml, equilibrated with the lysis buffer;QIAGEN), followed by stirring using a rotor (4° C. for 1 hour). Thecarrier was sunk by setting up the column and then the column was washedtwice using 4 ml for each of a wash buffer (50 mM NaH₂PO₄ (pH 8.0)containing 300 mM NaCl and 20 mM imidazole). Next, proteins were elutedby passing 4 times 0.5 ml for each of an elution buffer (50 mM NaH₂PO₄(pH 8.0) containing 300 mM NaCl and 250 mM imidazole). The eluatecontaining the protein of interest (1 ml) was dialyzed at 4° C. for 2days against 500 ml of PBS (phosphate buffered saline) containing 20%glycerol to thereby obtain about 0.5 ml (protein content 0.4 mg/ml) ofdialyzed solution (solution of the enzyme of the present invention(protein of the present invention)).

[0200] Western blotting of the thus obtained protein was carried out bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Inthe SDA-PAGE, 10% gel was used. Protein was detected by CoomassieBrilliant Blue staining. The Western blotting was carried out bytransferring protein in the SDS-PAGE gel onto a nitrocellulose membrane,blocking the membrane with 5% skimmed milk (dissolved in 25 mM Tris-HCl(pH 7.5) containing 150 mM NaCl and 0.05% Tween 20 (this solution is tobe called TBS-T)) and then treating it with anti-tetra-His antibody(Qiagen). After washing several times with TBS-T, this membrane wastreated with peroxidase-linked anti-mouse IgG. After washing with TBS-T,the reacted protein was detected by ECL detection system (Amersham).

[0201] As a result, the protein showed a band at around 80 kDa by theWestern blotting analysis using SDS-PAGE and anti-tetra-His antibody. Onthe other hand, an immunologically reacting band was not detected in theculture extract of a control (expression vector having no insert).

[0202] (2) Analysis of Enzyme Activity (Analysis of GalNAc TransferActivity)

[0203] The enzyme of the present invention (2 μg), hexasaccharide ofshark cartilage chondroitin sulfate C, purified by degrading withtesticular hyaluronidase, as the acceptor (70 pmol) and UDP-GalNAc (3nmol), UDP-GlcUA (3 nmol) and UDP-[³H]GalNAc (0.1 nmol, 0.1 μCi) as thedonors were added to 50 mM Tris-HCl (pH 7.2) containing 20 mM MnCl₂, 0.1M (NH₄)₂SO₄ and 1 Methylene glycol, and the total volume was adjusted to50 μl, and then the reaction was carried out at 30° C. for 30 minutesand the enzyme was heat-inactivated. To the reaction solution, 3 volumesof 95% ethanol containing 1.3% potassium acetate was added, and thesample was centrifuged at 10,000×g for 20 minutes. The precipitate wasdissolved in 50 μl of distilled water and applied to a Superdex Peptidecolumn (300×φ10 mm: Amersham Biosciences, chromatography conditions;buffer: 0.2 M NaCl, flow rate: 0.5 ml/min), and the eluate wasfractionated at 0.5 ml and the radioactivity (count of [³H]) of eachfraction was measured using a scintillation counter. Chondroitinsynthesizing activity was determined by calculating the amount ofradioactivity incorporated into fractions of higher molecular weightthan the acceptor substrate. The results are shown in FIG. 3. In FIG. 3,the closed squares indicate radioactivity when hexasaccharide ofchondroitin sulfate C was used as the acceptor, open triangles indicateradioactivity when the enzyme reaction product was treated withchondroitinase ABC and closed circles indicates control (whereinheat-inactivated enzyme of the present invention was used).

[0204] As a result, elution position of the radioactivity appeared at ahigher molecular weight side than the hexasaccharide of chondroitinsulfate C (the broad peak having its top at around 5,000 Da) (closedsquares in FIG. 3). Also, when the enzyme reaction product was treatedwith chondroitinase ABC, the high molecular weight peak was shifted to aposition corresponding to the disaccharide fraction (open triangles inFIG. 3). When disaccharide composition of this chondroitinase ABC digestwas analyzed using a high performance liquid chromatography (HPLC), onlyan un-sulfated, unsaturated chondroitin disaccharide (ΔdiOS) wasdetected.

[0205] Also, the enzyme reaction product was completely digested bychondroitinase ACII treatment too, but not digested by Streptomyceshyaluronidase and heparitinase I.

[0206] Based on the above, it was shown that the thus obtained enzyme ofthe present invention at least transfers GaINAc to the hexasaccharide ofchondroitin sulfate C from UDP-GalNAc (donor). This specific activitywas 3.25±0.64 nmol GalNAc/min/mg protein.

[0207] (3) Analysis of Size of Enzyme Reaction Product

[0208] The size of the enzyme reaction product was examined by carryingout the enzyme reaction and chromatography in the same manner as in theabove “(2) Analysis of enzyme activity”. The results are shown in FIG.4.

[0209] It was confirmed from FIG. 4 that the enzyme of the presentinvention obtained in the above at least transfers GalNAc to an acceptor(an oligosaccharide having the chondroitin backbone (hexasaccharide ofchondroitin sulfate C prepared using testicular hyaluronidase)) from aGalNAc donor (UDP-GalNAc) to thereby form chondroitin having a molecularweight of 10,000 to 20,000 or more.

[0210] (4) Analysis of Specificity of Donor Substrate

[0211] Using UDP-[¹⁴C]GlcUA, UDP-[¹⁴C]GlcNAc or UDP-[³H]GalNAc as thedonor, transferring, activity to the following acceptors was examined inaccordance with the method described in the above “(2) Enzyme activitymeasurement”. Products after the enzyme reaction were analyzed by gelfiltration.

[0212] Results of the use of hexasaccharide or heptasaccharide ofchondroitin sulfate C as the acceptor are shown in FIG. 5. Also, theclosed circle in FIG. 5 indicates a control (a case in whichheat-inactivated enzyme of the present invention was used).

[0213] (A) Hexasaccharide of chondroitin sulfate C (A product purifiedby degrading shark cartilage chondroitin sulfate C with testicularhyaluronidase, and the non-reduced terminal is GlcUA)

[0214] Heptasaccharide alone was synthesized when UDP-GalNAc alone wasused as the donor (closed lozenge in FIG. 5).

[0215] Substantial transfer was not found when UDP-GlcUA alone was usedas the donor (closed triangle in FIG. 5).

[0216] Although very little, transfer of GlcNAc was found whenUDP-GlcNAc alone was used as the donor. This transferring activity was6.3% based on 100% activity in the case of the use of UDP-GalNAc as thedonor (closed square in FIG. 5). However, polysaccharides having a sizeof octasaccharide or more were not obtained even when both of UDP-GlcNAcand UDP-GlcUA were used together with this.

[0217] In addition, since incorporation of the radioactivity was notfound in the absence of acceptor substrate, it was suggested that anacceptor substrate is essential for the synthesis of chondroitin by thisenzyme.

[0218] (B) Heptasaccharide of chondroitin sulfate C (A product obtainedby allowing the enzyme of the present invention to react with the abovehexasaccharide of chondroitin sulfate C and thereby linking one residueof GalNAc to the non-reduced terminal of the hexasaccharide)

[0219] Octasaccharide alone was synthesized when UDP-GlcNAc alone wasused as the donor (open triangle in FIG. 5).

[0220] When either UDP-GalNAc or UDP-GlcNAc alone was used as the donor,substantial transfer was not found in each case (respectively, openlozenge and open square in FIG. 5).

[0221] Also, results of carrying out similar tests each independentlyare shown in Table 1. Also, the term “CS” in Table 1 means chondroitinsulfate C. Also, parenthesis in the table shows length of sugar chainafter the enzyme reaction and “-” means that the labeled UDP-sugar wasnot transferred to the corresponding acceptor substrate. TABLE 1Chondroitin polymerase-specific activity (nmol/min/mg protein) Donorsubstrate Acceptor substrate Labeled Unlabeled Cs hepta- UDP-sugarUDP-sugar Cs hexasaccharide saccharide UDP-[³H]GalNAc none 0.59 ± 0.16(hepta)  0.0 ± 0.0  (−) UDP-[¹⁴C]GlcNAc none 0.04 ± 0.02 (hepta)  0.0 ±0.0  (−) UDP-[¹⁴C]GlcUA none  0.0 ± 0.0 (−) 0.53 ± 0.08 (octa)UDP-[³H]GalNAc UDP-GlcUA 3.25 ± 0.64 (poly) not measured UDP-[¹⁴C]GlcUAUDP-GalNAc 2.75 ± 0.28 (poly) not measured UDP-[¹⁴C]GlcNAc UDP-GlcUA0.05 ± 0.02 (poly) not measured UDP-[¹⁴C]GlcUA UDP-GlcNAc  0.0 ± 0.0 (−)not measured

[0222] From the above results, it was shown that the enzyme of thepresent invention obtained in the above transfers GalNAc from UDP-GalNActo a sugar chain (acceptor) having a chondroitin backbone whosenon-reduced terminal is GlcUA. Also, it was shown that when the acceptoris used, the enzyme of the present invention obtained in the above showsthe activity to transfer GlcNAc from UDP-GlcNAc, but the activity is farlower than its GalNAc transferring activity. Also, it was shown thatwhen the acceptor is used, the enzyme of the present invention obtainedin the above does not substantially have the activity to transfer GlcUAfrom UDP-GlcUA. Based on these results, it was shown that the aboveenzyme of the present invention is not capable of transferring GlcUA tothe non-reduced terminal GlcUA but is capable of transferring oneresidue of GalNAc (or, though slight, GlcNAc).

[0223] Also, it was shown that the enzyme of the present inventionobtained in the above transfers GlcUA from UDP-GlcUA to a sugar chain(acceptor) having a chondroitin backbone whose non-reduced terminal isGalNAc. Also, it was shown that when the acceptor is used, the enzyme ofthe present invention obtained in the above substantially have noactivities to transfer GalNAc from UDP-GalNAc and to transfer GlcNAcfrom UDP-GlcNAc. Based on these results, it was shown that the aboveenzyme of the present invention is not capable of transferring GalNAc tothe non-reduced terminal GalNAc but is capable of transferring oneresidue of GlcUA.

[0224] Based on the above, it was shown that the above enzyme of thepresent invention is capable of transferring GlcUA and GalNAcalternately from a GlcUA donor and a GalNAc donor, respectively, to thesugar chain non-reduced terminal.

[0225] (5) Analysis of Specificity of Acceptor Substrate

[0226] Using tetrasaccharide (140 pmol) or hexasaccharide (140 pmol) ofchondroitin sulfate C degraded and purified using testicularhyaluronidase, tetrasaccharide (260 pmol) or hexasaccharide (175 pmol)of chondroitin degraded and purified by the Nagasawa's method(Carbohydrate Research, 97, 263-278 (1981)), hexasaccharide (175 pmol)of hyaluronic acid degraded and purified using testicular hyaluronidase,chondroitin sulfate C (molecular weight 20,000), chondroitin (molecularweight 10,000), dermatan sulfate (molecular weight 15,000), hyaluronicacid (molecular weight 20,000) or heparin (molecular weight 10,000) asthe acceptor, the transferring activity was examined by the followingmethod. Also, the sugar chains were purchased from SeikagakuCorporation.

[0227] The enzyme of the present invention (2 μg), UDP-GalNAc (Sigma)(60 pmol), UDP-GlcUA (Sigma) (0.6 nmol) and UDP-[³H]GalNAc (0.1 nmol,0.1 μCi) as the donors and each of the above sugar chains as theacceptor were added to 50 mM Tris-HCl (pH 7.2) containing 20 mM MnCl₂,0.1 M (NH₄)₂SO₄ and 1 M ethylene glycol, and the total volume wasadjusted to 50 μl, and then the enzyme reaction was carried out at 30°C. for 30 minutes and the enzyme was heat-inactivated. The reactionsolution was applied to a Superdex Peptide column (300×φ10 mm: AmershamBiosciences, chromatography conditions; buffer: 0.2 M NaCl, flow rate:0.5 ml/min), and the eluate was fractionated at 0.5 ml and theradioactivity (Count of [³H]) of each fraction was measured using ascintillation counter. The results are shown in Table 2. Theparenthesized numerals in the table are relative values when thequantity of radioactivity (amount of transferred GalNAc) by the use ofthe hexasaccharide of chondroitin sulfate C as the acceptor was definedas 100%. TABLE 2 Specific activity of [³H] incorporation Acceptorsubstrate nmol/min/mg protein % Chondroitin Tetrasaccharide 1.44 ± 0.2443.0 sulfate C Hexasaccharide 3.34 ± 0.50 100.0 Polysaccharide 3.41 ±0.48 100.0 (M.W. 20,000) Chondroitin Tetrasaccharide 1.12 ± 0.21 33.5Hexasaccharide 1.24 ± 0.45 37.0 Polysaccharide 0.53 ± 0.13 15.8 (M.W.10,000) Hyaluronic acid Hexasaccharide 0.80 ± 0.15 24.0 Polysaccharide0.27 ± 0.02 8.2 (M.W. 20,000) Dermatan sulfate Polysaccharide 0.06 ±0.02 1.9 (M.W. 15,000) Heparin Polysaccharide 0.0 ± 0.0 0.0 (M.W.10,000)

[0228] Based on the above results, it was shown that the hexasaccharideof chondroitin sulfate C becomes the most suitable acceptor substrate.The chondroitin hexasaccharide also functioned as an acceptor substrate,but its activity was low (37%) in comparison with tho case of thehexasaccharide of chondroitin sulfate C. Incorporation into thechondroitin sulfate tetrasaccharide or chondroitin tetrasaccharide wasthe same (43% and 33.5%, respectively). To our surprise, the hyaluronicacid hexasaccharide and hyaluronic acid (molecular weight 20,000) alsofunctioned as acceptor substrates. The incorporation level ofchondroitin sulfate C (molecular weight 20,000) was similar to that ofthe hexasaccharide of chondroitin sulfate C. The incorporation was notso high in the case of chondroitin (molecular weight 10,000).

[0229] In summing up the above results, it was shown that the aboveenzyme of the present invention uses oligosaccharides andpolysaccharides having chondroitin backbone (at least tetrasaccharide,hexasaccharide and heptasaccharide of chondroitin sulfate C, chondroitintetrasaccharide and hexasaccharide, chondroitin sulfate C (molecularweight 20,000) and chondroitin (molecular weight 10,000)) andoligosaccharides and polysaccharides of hyaluronic acid (at leasthyaluronic acid hexasaccharide and hyaluronic acid (molecular weight20,000)) as acceptors.

[0230] On the other hand, incorporation of the radioactivity was notfound in dermatan sulfate (molecular weight 15,000) and heparin(molecular weight 10,000), showing that they do not substantiallyfunction as acceptor substrates.

[0231] (6) Analysis of Influence by Temperature

[0232] The enzyme reaction was carried out in the same manner as theabove “(2) Analysis of enzyme activity” by changing the enzyme reactiontemperature to 25, 30, 37, 40, 45 or 100° C., and the solution after thereaction was applied to a Superdex 75 column (300×φ10 mm: AmershamBiosciences, chromatography conditions; buffer: 0.2 M NaCl, flow rate:0.5 ml/min). The eluate was fractionated in 1 ml portions and theradioactivity (count of [³H]) of each fraction was measured using ascintillation counter. The results are shown in FIG. 6. Also, thelozenge, square, triangle, × and * marks in FIG. 6 show the results of25, 30, 37, 40, 45 and 100° C., respectively. Also, the circle in FIG. 6shows the result of a control (wherein heat-inactivated enzyme of thepresent invention was used).

[0233] As shown in FIG. 6, under the reaction conditions and within thetemperature range examined this time, the molecular weight of thereaction product became small as the temperature was increased. Thehighest incorporation was found at 30° C., but the product having thelargest molecular weight was obtained at 25° C.

[0234] It is considered from the results that enzyme activity of theabove enzyme of the present invention decreases as the reactiontemperature increases starting at 25° C. under the reaction conditionsof this time.

[0235] (7) Analysis of Influence by Metal Ions and the Like

[0236] The enzyme reaction was carried out in the same manner as theabove “(2) Analysis of enzyme activity”, except that each of variousmetal salts (MnCl₂, FeCl₂, MgCl₂, CaCl₂ or CuCl₂) or EDTA was addedinstead of MnCl₂, and the solution after the reaction was applied to aSuperdex 75 column (300×φ10 mm: Amersham Biosciences, chromatographyconditions; buffer: 0.2 M NaCl, flow rate: 0.5 ml/min). The eluate wasfractionated at 1 ml and the radioactivity (count of [³H]) of eachfraction was measured using a scintillation counter. Relative valueswhen the radioactivity in addition of MnCl₂ was defined as 100% areshown below. TABLE 3 Metal salt Relative value of radioactivity (%)MnCl₂ 100.0 FeCl₂ 30.6 MgCl₂ 30.7 CaCl₂ 0.0 CuCl₂ 0.0 EDTA 0.0

[0237] Based on the results, the above enzyme of the present inventionshowed the highest activity in the presence of Mn²⁺ ion. Also, in thepresence of Fe²⁺ or Mg²⁺ ion, it showed about 30% of the activity incomparison with the case of the presence of Mn²⁺ ion. Also, it was shownthat it does not substantially act in the presence of Ca²⁺ or Cu²⁺ ionor ethylenediaminetetraacetic acid.

[0238] In addition, it was shown that the above enzyme of the presentinvention acts in the presence of Fe²⁺ or Mg²⁺ ion too, and its enzymeactivity in the presence of Mn²⁺ ion is higher than the enzyme activityin the presence of Fe²⁺ or Mg^(2+ ion.)

[0239] (8) Optimum Reaction pH

[0240] When optimum reaction pH of the enzyme of the present inventionwas examined by changing the pH of the above “(2) Analysis of enzymeactivity” to various levels, it was from pH 7.0 to 7.5.

[0241] (9) Relation with Enzyme Reaction Time

[0242] By setting the enzyme reaction time to 10 minutes, 30 minutes, 1hour, 3 hours, 6 hours or 18 hours, the enzyme reaction was carried outin the same manner as the above “(2) Analysis of enzyme activity”, andthe radioactivity incorporated into the enzyme reaction product wasanalyzed. Gel filtration patterns of [³H]GalNAc after various reactionperiods are shown in FIG. 7, and the total amounts of the incorporationof radioactivity after various enzyme reaction periods in FIG. 8. Theopen circle, closed circle, open triangle, closed triangle, open squareand closed square show the results after 10 minutes, 30 minutes, 1 hour,3 hours, 6 hours and 18 hours, respectively. Also, the arrow with “20k”,“10k”, “5k”, “14”, “8” or “6” shows the elution position of molecularweight 20,000, 10,000, 5,000, tetradecasaccharide (molecular weight:about 2,800), octasaccharide (molecular weight: about 1,600) orhexasaccharide (molecular weight: about 1,200) of hyaluronic acid(standard), respectively.

[0243] It was shown from the results of FIG. 8 that under the testconditions, quick incorporation of [³H]GalNAc is found after 3 hours and6 hours, but the incorporation becomes slow as the reaction draws closeto 20 hours.

[0244] Also, from the results of FIG. 7, it was shown that theincorporation increases and a reaction product of high molecular weightis obtained after a long period of reaction time.

[0245] In addition, a high molecular weight product was quickly obtainedwhen an acceptor substrate (hexasaccharide) was set to a lowerconcentration, and a low molecular weight product was obtained when theacceptor substrate (hexasaccharide) was set to a high concentration.

[0246] (10) Determination of Michaelis Constant (Km)

[0247] The radioactivity incorporated into the enzyme reaction productwas measured in accordance with the above “(2) Analysis of enzymeactivity”, by setting using amount of the enzyme of the presentinvention to 1.3 μg, containing the one donor substrate (UDP-sugar;UDP-GlcUA or UDP-GalNAc) in a fixed concentration (240 μM), and addingthereto the other radio-labeled donor substrate (radiation UDP-sugar;UDP-[³H]GalNAc or UDP-[¹⁴C]GlcUA) having various concentrations (0.6 to200 μM). Independent tests were carried out three times, and the averagevalue was used as the measured value.

[0248] Relationship between the incorporated radioactivity (V) andsubstrate concentration (S) of UDP-sugar is shown in FIG. 9, and itsdouble reciprocal plot in FIG. 10. Closed circle and open square in thedrawings show results on UDP-GlcUA and UDP-GalNAc, respectively.

[0249] As the result, the Km for UDP-GlcUA and the Km for UDP-GalNAcwere calculated to be 3.44 μM and 31.6 μM, respectively.

[0250] While the present invention has been described in detail and withreference to specific embodiments thereof, it will be apparent to one ofskill in the art that various changes and modifications can be madetherein without departing from the spirit and scope thereof. Allreferences cited herein are incorporated in their entirety.

[0251] This application is based on Japanese application Nos.2001-244685, 2001-324127 and 2002-103136 filed on Aug. 10, 2001, Oct.22, 2001 and Apr. 5, 2002, the entire contents of which are incorporatedhereinto by reference.

1 12 1 2058 DNA Escherichia coli CDS (1)..(2058) 1 atg agt att ctt aatcaa gca ata aat tta tat aaa aac aaa aat tat 48 Met Ser Ile Leu Asn GlnAla Ile Asn Leu Tyr Lys Asn Lys Asn Tyr 1 5 10 15 cgc caa gct tta tctctt ttt gag aag gtt gct gaa att tat gat gtt 96 Arg Gln Ala Leu Ser LeuPhe Glu Lys Val Ala Glu Ile Tyr Asp Val 20 25 30 agt tgg gtc gaa gca aatata aaa tta tgc caa acc gca ctc aat ctt 144 Ser Trp Val Glu Ala Asn IleLys Leu Cys Gln Thr Ala Leu Asn Leu 35 40 45 tct gaa gaa gtt gat aag ttaaat cgt aaa gct gtt att gat att gat 192 Ser Glu Glu Val Asp Lys Leu AsnArg Lys Ala Val Ile Asp Ile Asp 50 55 60 gca gca aca aaa ata atg tgt tctaac gcc aaa gca att agt ctg aac 240 Ala Ala Thr Lys Ile Met Cys Ser AsnAla Lys Ala Ile Ser Leu Asn 65 70 75 80 gag gtt gaa aaa aat gaa ata ataagc aaa tac cga gaa ata acc gca 288 Glu Val Glu Lys Asn Glu Ile Ile SerLys Tyr Arg Glu Ile Thr Ala 85 90 95 aag aaa tca gaa cgg gcg gag tta aaggaa gtc gaa ccc att cct tta 336 Lys Lys Ser Glu Arg Ala Glu Leu Lys GluVal Glu Pro Ile Pro Leu 100 105 110 gat tgg cct agt gat tta act tta ccgccg tta cct gag agc aca aac 384 Asp Trp Pro Ser Asp Leu Thr Leu Pro ProLeu Pro Glu Ser Thr Asn 115 120 125 gat tat gtt tgg gcg ggg aaa aga aaagag ctt gat gat tat cca aga 432 Asp Tyr Val Trp Ala Gly Lys Arg Lys GluLeu Asp Asp Tyr Pro Arg 130 135 140 aaa cag tta atc att gac ggg ctt agtatt gta att cct aca tat aat 480 Lys Gln Leu Ile Ile Asp Gly Leu Ser IleVal Ile Pro Thr Tyr Asn 145 150 155 160 cga gca aaa ata ctt gca att acactt gct tgt ctt tgt aac caa aag 528 Arg Ala Lys Ile Leu Ala Ile Thr LeuAla Cys Leu Cys Asn Gln Lys 165 170 175 acc ata tac gac tat gaa gtt attgtt gcc gat gat gga agt aaa gaa 576 Thr Ile Tyr Asp Tyr Glu Val Ile ValAla Asp Asp Gly Ser Lys Glu 180 185 190 aat att gaa gaa ata gta aga gaattt gaa agt tta tta aat ata aaa 624 Asn Ile Glu Glu Ile Val Arg Glu PheGlu Ser Leu Leu Asn Ile Lys 195 200 205 tat gta cgt cag aag gat tat ggatat caa ctg tgt gct gtt aga aat 672 Tyr Val Arg Gln Lys Asp Tyr Gly TyrGln Leu Cys Ala Val Arg Asn 210 215 220 ctt ggg ctt agg gct gca aag tataat tat gtt gca att ctg gat tgt 720 Leu Gly Leu Arg Ala Ala Lys Tyr AsnTyr Val Ala Ile Leu Asp Cys 225 230 235 240 gat atg gct ccg aac cca ctatgg gtt cag tca tat atg gaa cta tta 768 Asp Met Ala Pro Asn Pro Leu TrpVal Gln Ser Tyr Met Glu Leu Leu 245 250 255 gcg gtg gac gat aat gtt gctcta att ggc cct aga aaa tat ata gat 816 Ala Val Asp Asp Asn Val Ala LeuIle Gly Pro Arg Lys Tyr Ile Asp 260 265 270 aca agc aag cat aca tat ttagat ttc ctt tcc caa aaa tca cta ata 864 Thr Ser Lys His Thr Tyr Leu AspPhe Leu Ser Gln Lys Ser Leu Ile 275 280 285 aat gaa att cct gaa atc attact aat aat cag gtt gca ggc aag gtt 912 Asn Glu Ile Pro Glu Ile Ile ThrAsn Asn Gln Val Ala Gly Lys Val 290 295 300 gag caa aac aaa tca gtt gactgg cga ata gaa cat ttc aaa aat acc 960 Glu Gln Asn Lys Ser Val Asp TrpArg Ile Glu His Phe Lys Asn Thr 305 310 315 320 gat aat cta aga tta tgcaac aca cca ttt cga ttt ttt agc gga ggt 1008 Asp Asn Leu Arg Leu Cys AsnThr Pro Phe Arg Phe Phe Ser Gly Gly 325 330 335 aat gtc gct ttt gcg aaaaaa tgg ctt ttc cgt gca gga tgg ttt gat 1056 Asn Val Ala Phe Ala Lys LysTrp Leu Phe Arg Ala Gly Trp Phe Asp 340 345 350 gaa gag ttt acg cat tggggg ggg gag gat aat gag ttt gga tat cgt 1104 Glu Glu Phe Thr His Trp GlyGly Glu Asp Asn Glu Phe Gly Tyr Arg 355 360 365 ctc tac aga gaa gga tgttac ttt cgg tct gtt gaa gga gca atg gca 1152 Leu Tyr Arg Glu Gly Cys TyrPhe Arg Ser Val Glu Gly Ala Met Ala 370 375 380 tat cat caa gaa cca cccggg aaa gaa aac gag acg gat cgt gcg gca 1200 Tyr His Gln Glu Pro Pro GlyLys Glu Asn Glu Thr Asp Arg Ala Ala 385 390 395 400 ggg aaa aat att actgtt caa ttg tta cag caa aaa gtt cct tat ttc 1248 Gly Lys Asn Ile Thr ValGln Leu Leu Gln Gln Lys Val Pro Tyr Phe 405 410 415 tat aga aaa aaa gaaaaa ata gaa tcc gcg aca tta aaa aga gta cca 1296 Tyr Arg Lys Lys Glu LysIle Glu Ser Ala Thr Leu Lys Arg Val Pro 420 425 430 cta gta tct ata tatatt ccc gcc tat aac tgc tct aaa tat att gtt 1344 Leu Val Ser Ile Tyr IlePro Ala Tyr Asn Cys Ser Lys Tyr Ile Val 435 440 445 cgt tgt gtt gaa agcgcc ctt aat cag aca ata act gac tta gaa gta 1392 Arg Cys Val Glu Ser AlaLeu Asn Gln Thr Ile Thr Asp Leu Glu Val 450 455 460 tgc ata tgc gat gatggt tcc aca gat gat aca ttg cgg att ctt cag 1440 Cys Ile Cys Asp Asp GlySer Thr Asp Asp Thr Leu Arg Ile Leu Gln 465 470 475 480 gag cat tat gcaaac cat cct cga gtt cgt ttt att tca caa aaa aac 1488 Glu His Tyr Ala AsnHis Pro Arg Val Arg Phe Ile Ser Gln Lys Asn 485 490 495 aaa gga att ggttca gca tct aat aca gca gtt aga ttg tgt cgg gga 1536 Lys Gly Ile Gly SerAla Ser Asn Thr Ala Val Arg Leu Cys Arg Gly 500 505 510 ttc tat ata ggtcag tta gac tct gat gac ttt ctt gaa cca gat gct 1584 Phe Tyr Ile Gly GlnLeu Asp Ser Asp Asp Phe Leu Glu Pro Asp Ala 515 520 525 gtt gaa cta tgtcta gat gaa ttt aga aaa gat cta tca ttg gca tgt 1632 Val Glu Leu Cys LeuAsp Glu Phe Arg Lys Asp Leu Ser Leu Ala Cys 530 535 540 gtt tat aca actaac cgt aat ata gat cgt gaa ggt aat ttg ata tca 1680 Val Tyr Thr Thr AsnArg Asn Ile Asp Arg Glu Gly Asn Leu Ile Ser 545 550 555 560 aat ggc tataat tgg ccc att tat tcg cga gaa aaa ctt act agt gca 1728 Asn Gly Tyr AsnTrp Pro Ile Tyr Ser Arg Glu Lys Leu Thr Ser Ala 565 570 575 atg ata tgtcat cat ttc agg atg ttc aca gca aga gca tgg aac cta 1776 Met Ile Cys HisHis Phe Arg Met Phe Thr Ala Arg Ala Trp Asn Leu 580 585 590 act gaa ggtttc aac gaa tcg atc agc aac gca gtt gat tac gat atg 1824 Thr Glu Gly PheAsn Glu Ser Ile Ser Asn Ala Val Asp Tyr Asp Met 595 600 605 tat tta aaactt agt gaa gtt gga ccg ttc aag cat ata aac aaa att 1872 Tyr Leu Lys LeuSer Glu Val Gly Pro Phe Lys His Ile Asn Lys Ile 610 615 620 tgt tat aatcgc gta ttg cat ggt gaa aat acg tct ata aaa aag ttg 1920 Cys Tyr Asn ArgVal Leu His Gly Glu Asn Thr Ser Ile Lys Lys Leu 625 630 635 640 gat attcaa aag gaa aat cat ttt aaa gtt gtt aac gaa tca tta agt 1968 Asp Ile GlnLys Glu Asn His Phe Lys Val Val Asn Glu Ser Leu Ser 645 650 655 agg ctaggc ata aaa aaa tat aaa tat tca cca tta act aat ttg aat 2016 Arg Leu GlyIle Lys Lys Tyr Lys Tyr Ser Pro Leu Thr Asn Leu Asn 660 665 670 gaa tgtaga aaa tat acc tgg gaa aaa ata gag aat gat tta 2058 Glu Cys Arg Lys TyrThr Trp Glu Lys Ile Glu Asn Asp Leu 675 680 685 2 686 PRT Escherichiacoli 2 Met Ser Ile Leu Asn Gln Ala Ile Asn Leu Tyr Lys Asn Lys Asn Tyr 15 10 15 Arg Gln Ala Leu Ser Leu Phe Glu Lys Val Ala Glu Ile Tyr Asp Val20 25 30 Ser Trp Val Glu Ala Asn Ile Lys Leu Cys Gln Thr Ala Leu Asn Leu35 40 45 Ser Glu Glu Val Asp Lys Leu Asn Arg Lys Ala Val Ile Asp Ile Asp50 55 60 Ala Ala Thr Lys Ile Met Cys Ser Asn Ala Lys Ala Ile Ser Leu Asn65 70 75 80 Glu Val Glu Lys Asn Glu Ile Ile Ser Lys Tyr Arg Glu Ile ThrAla 85 90 95 Lys Lys Ser Glu Arg Ala Glu Leu Lys Glu Val Glu Pro Ile ProLeu 100 105 110 Asp Trp Pro Ser Asp Leu Thr Leu Pro Pro Leu Pro Glu SerThr Asn 115 120 125 Asp Tyr Val Trp Ala Gly Lys Arg Lys Glu Leu Asp AspTyr Pro Arg 130 135 140 Lys Gln Leu Ile Ile Asp Gly Leu Ser Ile Val IlePro Thr Tyr Asn 145 150 155 160 Arg Ala Lys Ile Leu Ala Ile Thr Leu AlaCys Leu Cys Asn Gln Lys 165 170 175 Thr Ile Tyr Asp Tyr Glu Val Ile ValAla Asp Asp Gly Ser Lys Glu 180 185 190 Asn Ile Glu Glu Ile Val Arg GluPhe Glu Ser Leu Leu Asn Ile Lys 195 200 205 Tyr Val Arg Gln Lys Asp TyrGly Tyr Gln Leu Cys Ala Val Arg Asn 210 215 220 Leu Gly Leu Arg Ala AlaLys Tyr Asn Tyr Val Ala Ile Leu Asp Cys 225 230 235 240 Asp Met Ala ProAsn Pro Leu Trp Val Gln Ser Tyr Met Glu Leu Leu 245 250 255 Ala Val AspAsp Asn Val Ala Leu Ile Gly Pro Arg Lys Tyr Ile Asp 260 265 270 Thr SerLys His Thr Tyr Leu Asp Phe Leu Ser Gln Lys Ser Leu Ile 275 280 285 AsnGlu Ile Pro Glu Ile Ile Thr Asn Asn Gln Val Ala Gly Lys Val 290 295 300Glu Gln Asn Lys Ser Val Asp Trp Arg Ile Glu His Phe Lys Asn Thr 305 310315 320 Asp Asn Leu Arg Leu Cys Asn Thr Pro Phe Arg Phe Phe Ser Gly Gly325 330 335 Asn Val Ala Phe Ala Lys Lys Trp Leu Phe Arg Ala Gly Trp PheAsp 340 345 350 Glu Glu Phe Thr His Trp Gly Gly Glu Asp Asn Glu Phe GlyTyr Arg 355 360 365 Leu Tyr Arg Glu Gly Cys Tyr Phe Arg Ser Val Glu GlyAla Met Ala 370 375 380 Tyr His Gln Glu Pro Pro Gly Lys Glu Asn Glu ThrAsp Arg Ala Ala 385 390 395 400 Gly Lys Asn Ile Thr Val Gln Leu Leu GlnGln Lys Val Pro Tyr Phe 405 410 415 Tyr Arg Lys Lys Glu Lys Ile Glu SerAla Thr Leu Lys Arg Val Pro 420 425 430 Leu Val Ser Ile Tyr Ile Pro AlaTyr Asn Cys Ser Lys Tyr Ile Val 435 440 445 Arg Cys Val Glu Ser Ala LeuAsn Gln Thr Ile Thr Asp Leu Glu Val 450 455 460 Cys Ile Cys Asp Asp GlySer Thr Asp Asp Thr Leu Arg Ile Leu Gln 465 470 475 480 Glu His Tyr AlaAsn His Pro Arg Val Arg Phe Ile Ser Gln Lys Asn 485 490 495 Lys Gly IleGly Ser Ala Ser Asn Thr Ala Val Arg Leu Cys Arg Gly 500 505 510 Phe TyrIle Gly Gln Leu Asp Ser Asp Asp Phe Leu Glu Pro Asp Ala 515 520 525 ValGlu Leu Cys Leu Asp Glu Phe Arg Lys Asp Leu Ser Leu Ala Cys 530 535 540Val Tyr Thr Thr Asn Arg Asn Ile Asp Arg Glu Gly Asn Leu Ile Ser 545 550555 560 Asn Gly Tyr Asn Trp Pro Ile Tyr Ser Arg Glu Lys Leu Thr Ser Ala565 570 575 Met Ile Cys His His Phe Arg Met Phe Thr Ala Arg Ala Trp AsnLeu 580 585 590 Thr Glu Gly Phe Asn Glu Ser Ile Ser Asn Ala Val Asp TyrAsp Met 595 600 605 Tyr Leu Lys Leu Ser Glu Val Gly Pro Phe Lys His IleAsn Lys Ile 610 615 620 Cys Tyr Asn Arg Val Leu His Gly Glu Asn Thr SerIle Lys Lys Leu 625 630 635 640 Asp Ile Gln Lys Glu Asn His Phe Lys ValVal Asn Glu Ser Leu Ser 645 650 655 Arg Leu Gly Ile Lys Lys Tyr Lys TyrSer Pro Leu Thr Asn Leu Asn 660 665 670 Glu Cys Arg Lys Tyr Thr Trp GluLys Ile Glu Asn Asp Leu 675 680 685 3 14483 DNA Escherichia coli CDS(3787)..(5847) 3 cgacattatg tctttaagaa tttaaatatt gaaatccctt caggaaaaagtgttgccttt 60 attggtcgta atggtgcggg taaatcaacg ttactgagaa tgattggtggcattgaccgc 120 cccgatagcg gaaagatcat caccaataaa acgatatcat ggccagtcggccttgcaggt 180 ggatttcagg gaagtttaac cggacgcgaa aatgtaaaat ttgtcgcgaggttatacgcg 240 aagcaagaag aactgaaaga gaaaattgag tttgttgaag aatttgccgaactcggcaag 300 tattttgata tgccgatcaa aacttactcc tctggtatgc gatctcgcctaggctttggt 360 ttaagtatgg catttaaatt tgattattat atcgtcgatg aagtaaccgcagtcggtgat 420 gccaggttta aagaaaaatg cgctcaattg tttaaagaaa ggcataaagaatctagtttt 480 ttaatggttt cacatagttt gaattcattg aaagagtttt gtgatgtggccattgttttt 540 aaggatgaca atgcggttag ttttcatgag gatgttcagg aggggatagaagagtatata 600 acggaacaaa ataattactg atgttgtttt caagggtgaa aaatgaatatattagttaca 660 ggtggagcag gctatattgg ctcgcatact agtttatgtc ttctgaataaaggttacaat 720 gttgtaatca ttgacaactt aattaattca tcttgcgaga gcattcgaaggattgaatta 780 atagctaaaa aaaaagttac tttctatgag ttgaacatca acaatgaaaaagaagttaat 840 caaattctaa aaaaacacaa atttgattgt ataatgcatt ttgccggtgcaaagtctgtt 900 gctgaatctt taataaaacc cattttttat tatgataata atgtttcagggacgttgcaa 960 ttaattaatt gcgctataaa aaacgatgtg gctaatttta tttttagctcttctgcaacg 1020 gtttatggtg aaagcaaaat aatgcctgta acagaagatt gccatataggaggaacatta 1080 aatccatatg gtacatcaaa gtatatatca gaattgatga ttagagatattgcaaaaaaa 1140 tatagcgata ctaatttttt gtgtctgaga tattttaacc caacaggtgctcacgagtcg 1200 ggaatgatcg gtgaaagtcc cgctgatata ccaagcaatt tagttccttatatattacaa 1260 gttgctatgg gtaaactaga aaaacttatg gtgtttgggg gggattaccctacaaaggat 1320 ggaaccggtg ttcgtgatta tatacacgta atggatttag cggaagggcatgtggctgct 1380 ttatcttacc ttttccgtga taataacact aattatcatg tttttaatttaggtactggt 1440 aaaggatatt ctgttttaga gctggtttct acctttgaaa aaatatctggggttagaatt 1500 ccatatgaaa ttgtttcgag aagagatggg gatattgctg aaagttggtcatcaccagaa 1560 aaagcaaata agtatctcaa ttggaaagct aaaagggaat tggaaacaatgcttgaggat 1620 gcctggcgct ggcaaatgaa aaacccaaat ggttatattt aatcatgcataaggtaaagc 1680 aaaattttac gataacttct atatctattg gcatcttaat aatcgaaatttagtgtggtg 1740 aattggttta ataaattcgt agacactaaa aagtagatgg ggtgttaattaaagctaact 1800 aaacaaatgg cgaataaagg gttttctgcc ttggggggat aaaacatcatgaatagacta 1860 gtaatagttg gtcatccgag ctctaattat caaattgtag aagaacttttgcatcaaaga 1920 ggaatgaatt ctctatgtcc atcgaagcga gataatttaa gcccccaagatatcactcag 1980 acgcttcgta aggcatatca atcccctgat atatacactg taacagatagtgctgatttc 2040 gaaccattac acgtttctac tgtctggaat ggtatagccc ttgacttgatgcttagtaat 2100 ttgaaccaaa aattgtgcgg atggtcggat cctaatgcaa tccatacattagaatattgg 2160 aagagtgttg atgaaaacat tacatttatt ctaatttacg atcatccaaagtctatttta 2220 acaaattatt tttcagatca aaatatatcg tccaactata cgtcagaacatttaattaaa 2280 aactggcttg cctataatac agcattgtta cacttttttc ttaataatcgcggtaggtgt 2340 ttattagtaa gctcagaaca agtcaaacgt aatgcagagg attgcatacaacaacttcaa 2400 cataagctta agttgaaatt tggtctttca ttttcaaata cgattaatcattcactagaa 2460 caatctgtaa atgattttaa gacggctgaa gcttcgatta ctctggaaaaagagcatcag 2520 gaaataatgt ctctatcagg tattgatata ggaaccggag atattatattcaaacagagt 2580 gaaacagagg aatatttaat tttcaatgta ttgaatgatt atccagattgtaaagagctt 2640 tattttgaat tacaatctaa tgcaaatact ccgcttaggg ttttagaaaaggaaaattac 2700 aagccttcct ttatatggga gacatttata aaacaacgcc aaataacattagatattgtc 2760 aatggattat atcagtcctc taaaaaaata attttagata acgagttacacacatcaaaa 2820 caattaaatg catatcaggc tattttaaaa gaattgagtg actctaaagaagaattgatt 2880 caatatgatt taataataaa aaataagaca atacaagttc aggagcttgaatgcgccata 2940 gaaaattttg aatcgctgtt aaagaaagaa caaaataaaa atgaattgcaacaacaaaga 3000 ttggaaaaat taagttgtga aaaagaacta ctgcttaatc aattgcatttagtacaacaa 3060 aaacttgaac aatattttat tgataatcaa cgattagaaa aaaaacaacttccagaatta 3120 tatggtgcag cagaacgtat aacacaggac attggatatc gcctgggtgctgtaatggtt 3180 agtcgttcaa aaacattttt aggattaatt agcattcctt ttgctttgatatccgaatgg 3240 cgaacatgga aaaagaaata cgatagtgaa tatcaagtct ctctgccatcaatatttctt 3300 tatgctgata aacatgaggc agaaagggtg aaaaaacatt tatcctatcaattaggaaaa 3360 ttaatcataa atcaaaatca ttttccacta gggttgatat ctttgccattttcgatatac 3420 agaacaatac gtcaattcaa aagaacaaaa aataattctc aggtaggggttaaatactgt 3480 ggaaaataaa tccaggctac taaatataaa gttaaaatat cggctataaatgcgtgcgaa 3540 ttaatagtga aaattttctt agttaagtga aatagctttt ttctaattgtttaagtcata 3600 gtggtttacg ctttatttaa ttaaaaaaat aaaataataa attaaaaatacgattctcaa 3660 tatttcttca agtatcaatt aggatttaat ggggcaagat tatgatatcgcatgaaaata 3720 tatatatagg gacatgatta ttatgcgtgt tgatacttta attatactagattaggttga 3780 aataat atg agt att ctt aat caa gca ata aat tta tat aaaaac aaa 3828 Met Ser Ile Leu Asn Gln Ala Ile Asn Leu Tyr Lys Asn Lys 1 510 aat tat cgc caa gct tta tct ctt ttt gag aag gtt gct gaa att tat 3876Asn Tyr Arg Gln Ala Leu Ser Leu Phe Glu Lys Val Ala Glu Ile Tyr 15 20 2530 gat gtt agt tgg gtc gaa gca aat ata aaa tta tgc caa acc gca ctc 3924Asp Val Ser Trp Val Glu Ala Asn Ile Lys Leu Cys Gln Thr Ala Leu 35 40 45aat ctt tct gaa gaa gtt gat aag tta aat cgt aaa gct gtt att gat 3972 AsnLeu Ser Glu Glu Val Asp Lys Leu Asn Arg Lys Ala Val Ile Asp 50 55 60 attgat gca gca aca aaa ata atg tgt tct aac gcc aaa gca att agt 4020 Ile AspAla Ala Thr Lys Ile Met Cys Ser Asn Ala Lys Ala Ile Ser 65 70 75 ctg aacgag gtt gaa aaa aat gaa ata ata agc aaa tac cga gaa ata 4068 Leu Asn GluVal Glu Lys Asn Glu Ile Ile Ser Lys Tyr Arg Glu Ile 80 85 90 acc gca aagaaa tca gaa cgg gcg gag tta aag gaa gtc gaa ccc att 4116 Thr Ala Lys LysSer Glu Arg Ala Glu Leu Lys Glu Val Glu Pro Ile 95 100 105 110 cct ttagat tgg cct agt gat tta act tta ccg ccg tta cct gag agc 4164 Pro Leu AspTrp Pro Ser Asp Leu Thr Leu Pro Pro Leu Pro Glu Ser 115 120 125 aca aacgat tat gtt tgg gcg ggg aaa aga aaa gag ctt gat gat tat 4212 Thr Asn AspTyr Val Trp Ala Gly Lys Arg Lys Glu Leu Asp Asp Tyr 130 135 140 cca agaaaa cag tta atc att gac ggg ctt agt att gta att cct aca 4260 Pro Arg LysGln Leu Ile Ile Asp Gly Leu Ser Ile Val Ile Pro Thr 145 150 155 tat aatcga gca aaa ata ctt gca att aca ctt gct tgt ctt tgt aac 4308 Tyr Asn ArgAla Lys Ile Leu Ala Ile Thr Leu Ala Cys Leu Cys Asn 160 165 170 caa aagacc ata tac gac tat gaa gtt att gtt gcc gat gat gga agt 4356 Gln Lys ThrIle Tyr Asp Tyr Glu Val Ile Val Ala Asp Asp Gly Ser 175 180 185 190 aaagaa aat att gaa gaa ata gta aga gaa ttt gaa agt tta tta aat 4404 Lys GluAsn Ile Glu Glu Ile Val Arg Glu Phe Glu Ser Leu Leu Asn 195 200 205 ataaaa tat gta cgt cag aag gat tat gga tat caa ctg tgt gct gtt 4452 Ile LysTyr Val Arg Gln Lys Asp Tyr Gly Tyr Gln Leu Cys Ala Val 210 215 220 agaaat ctt ggg ctt agg gct gca aag tat aat tat gtt gca att ctg 4500 Arg AsnLeu Gly Leu Arg Ala Ala Lys Tyr Asn Tyr Val Ala Ile Leu 225 230 235 gattgt gat atg gct ccg aac cca cta tgg gtt cag tca tat atg gaa 4548 Asp CysAsp Met Ala Pro Asn Pro Leu Trp Val Gln Ser Tyr Met Glu 240 245 250 ctatta gcg gtg gac gat aat gtt gct cta att ggc cct aga aaa tat 4596 Leu LeuAla Val Asp Asp Asn Val Ala Leu Ile Gly Pro Arg Lys Tyr 255 260 265 270ata gat aca agc aag cat aca tat tta gat ttc ctt tcc caa aaa tca 4644 IleAsp Thr Ser Lys His Thr Tyr Leu Asp Phe Leu Ser Gln Lys Ser 275 280 285cta ata aat gaa att cct gaa atc att act aat aat cag gtt gca ggc 4692 LeuIle Asn Glu Ile Pro Glu Ile Ile Thr Asn Asn Gln Val Ala Gly 290 295 300aag gtt gag caa aac aaa tca gtt gac tgg cga ata gaa cat ttc aaa 4740 LysVal Glu Gln Asn Lys Ser Val Asp Trp Arg Ile Glu His Phe Lys 305 310 315aat acc gat aat cta aga tta tgc aac aca cca ttt cga ttt ttt agc 4788 AsnThr Asp Asn Leu Arg Leu Cys Asn Thr Pro Phe Arg Phe Phe Ser 320 325 330gga ggt aat gtc gct ttt gcg aaa aaa tgg ctt ttc cgt gca gga tgg 4836 GlyGly Asn Val Ala Phe Ala Lys Lys Trp Leu Phe Arg Ala Gly Trp 335 340 345350 ttt gat gaa gag ttt acg cat tgg ggg ggg gag gat aat gag ttt gga 4884Phe Asp Glu Glu Phe Thr His Trp Gly Gly Glu Asp Asn Glu Phe Gly 355 360365 tat cgt ctc tac aga gaa gga tgt tac ttt cgg tct gtt gaa gga gca 4932Tyr Arg Leu Tyr Arg Glu Gly Cys Tyr Phe Arg Ser Val Glu Gly Ala 370 375380 atg gca tat cat caa gaa cca ccc ggg aaa gaa aac gag acg gat cgt 4980Met Ala Tyr His Gln Glu Pro Pro Gly Lys Glu Asn Glu Thr Asp Arg 385 390395 gcg gca ggg aaa aat att act gtt caa ttg tta cag caa aaa gtt cct 5028Ala Ala Gly Lys Asn Ile Thr Val Gln Leu Leu Gln Gln Lys Val Pro 400 405410 tat ttc tat aga aaa aaa gaa aaa ata gaa tcc gcg aca tta aaa aga 5076Tyr Phe Tyr Arg Lys Lys Glu Lys Ile Glu Ser Ala Thr Leu Lys Arg 415 420425 430 gta cca cta gta tct ata tat att ccc gcc tat aac tgc tct aaa tat5124 Val Pro Leu Val Ser Ile Tyr Ile Pro Ala Tyr Asn Cys Ser Lys Tyr 435440 445 att gtt cgt tgt gtt gaa agc gcc ctt aat cag aca ata act gac tta5172 Ile Val Arg Cys Val Glu Ser Ala Leu Asn Gln Thr Ile Thr Asp Leu 450455 460 gaa gta tgc ata tgc gat gat ggt tcc aca gat gat aca ttg cgg att5220 Glu Val Cys Ile Cys Asp Asp Gly Ser Thr Asp Asp Thr Leu Arg Ile 465470 475 ctt cag gag cat tat gca aac cat cct cga gtt cgt ttt att tca caa5268 Leu Gln Glu His Tyr Ala Asn His Pro Arg Val Arg Phe Ile Ser Gln 480485 490 aaa aac aaa gga att ggt tca gca tct aat aca gca gtt aga ttg tgt5316 Lys Asn Lys Gly Ile Gly Ser Ala Ser Asn Thr Ala Val Arg Leu Cys 495500 505 510 cgg gga ttc tat ata ggt cag tta gac tct gat gac ttt ctt gaacca 5364 Arg Gly Phe Tyr Ile Gly Gln Leu Asp Ser Asp Asp Phe Leu Glu Pro515 520 525 gat gct gtt gaa cta tgt cta gat gaa ttt aga aaa gat cta tcattg 5412 Asp Ala Val Glu Leu Cys Leu Asp Glu Phe Arg Lys Asp Leu Ser Leu530 535 540 gca tgt gtt tat aca act aac cgt aat ata gat cgt gaa ggt aatttg 5460 Ala Cys Val Tyr Thr Thr Asn Arg Asn Ile Asp Arg Glu Gly Asn Leu545 550 555 ata tca aat ggc tat aat tgg ccc att tat tcg cga gaa aaa cttact 5508 Ile Ser Asn Gly Tyr Asn Trp Pro Ile Tyr Ser Arg Glu Lys Leu Thr560 565 570 agt gca atg ata tgt cat cat ttc agg atg ttc aca gca aga gcatgg 5556 Ser Ala Met Ile Cys His His Phe Arg Met Phe Thr Ala Arg Ala Trp575 580 585 590 aac cta act gaa ggt ttc aac gaa tcg atc agc aac gca gttgat tac 5604 Asn Leu Thr Glu Gly Phe Asn Glu Ser Ile Ser Asn Ala Val AspTyr 595 600 605 gat atg tat tta aaa ctt agt gaa gtt gga ccg ttc aag catata aac 5652 Asp Met Tyr Leu Lys Leu Ser Glu Val Gly Pro Phe Lys His IleAsn 610 615 620 aaa att tgt tat aat cgc gta ttg cat ggt gaa aat acg tctata aaa 5700 Lys Ile Cys Tyr Asn Arg Val Leu His Gly Glu Asn Thr Ser IleLys 625 630 635 aag ttg gat att caa aag gaa aat cat ttt aaa gtt gtt aacgaa tca 5748 Lys Leu Asp Ile Gln Lys Glu Asn His Phe Lys Val Val Asn GluSer 640 645 650 tta agt agg cta ggc ata aaa aaa tat aaa tat tca cca ttaact aat 5796 Leu Ser Arg Leu Gly Ile Lys Lys Tyr Lys Tyr Ser Pro Leu ThrAsn 655 660 665 670 ttg aat gaa tgt aga aaa tat acc tgg gaa aaa ata gagaat gat tta 5844 Leu Asn Glu Cys Arg Lys Tyr Thr Trp Glu Lys Ile Glu AsnAsp Leu 675 680 685 taa ttattgatat attacaagtg ataaacatgt agactggccccctgaatctc 5897 cagacaacca atatcactta aataagtgat agtcttaata ctagtttttagactagtcat 5957 tggagaacag atgattgatg tcttagggcc ggagaaacgc agacggcgtactacacagga 6017 aaagatcgct atcgttcagc agagctttga accgggaatg acggtctcccttgttgcccg 6077 gcaacatggt gtggcagcca gccagctatt tctctggcgt aagcaataccaggagggaag 6137 tcttactgct gtggctgccg gagaacaggt cgttcctgcc tctgaacttgctgccgccat 6197 gaagcagatt aaagaactcc agcgtctgct cggcaaaaaa acgatggaaaatgaactcct 6257 taaagaagcc gttgaatatg ggcgagcaaa aaagtggata gcgcacgcgcccttattgcc 6317 cggggatggg gagtaagctt cgtcagccgt tgtctccggg tgtcgcgtgcgcagttgcac 6377 gtcattctca gacgagccga tgactggaag gacggccgcc gcagccgtcacacggatgat 6437 acggatgtgc ttcgccgtat acaccatgtt atcggagagc tgcccacgtatggttatcgt 6497 cgggtatggg cgctgcttcg cagacaaaca gaacttgatg gtatgcctgcgatcaatgcc 6557 aaacgtgttt accggaccat gcgccagaat gcgctgttgc ttgagcgaaaacccgctgta 6617 ccgccatcga aacgggcaca taccggcaga gtggctgtga aagaaagtaatcagcgatgg 6677 tgctctgacg ggtttgagtt ccgctgtgat aacggagaaa aactgcgggtcacgttcgcg 6737 ctggactgct gtgaccgtga ggcactgcac tgggcggtca caacgggtggcttcgacagt 6797 gaaacagtac aggacgtcat gctgggagca gtggaacgcc gctttggcagcgagcttccg 6857 gcgtctccag tggagtggct gacggataat ggttcatgct accgggcgaatgaaacacgt 6917 cagttcgcca ggatgttggg acttgaaccg aagaacacgg cagtgcggagtccggagagt 6977 aacggaatag cagagagctt cgtgaaaacg ataaagcgtg actacataagtatcatgccc 7037 aaaccagacg ggttaacggc agcaaagaac cttgcagagg cgttcgagcattataacgaa 7097 tggcatccgc atagtgcgct gggttatcgc tcgccacggg aatatctgcggcagtgggcc 7157 agtgatgggt taagtgataa caggtatctg gaaacatagg ggcaaatccaacatgctaaa 7217 aaacttaaca tttgatcata tattaagtct ttcaaagaaa gaagataaaattaaacttgt 7277 acaattaatt gtaaatcatt tagatgagag aacattaagc tgtataaaaaatatctctac 7337 tggtaaggga tttaatgctc atttaaaaat acttgaactt tttgacctatggttgagtga 7397 gtattttgaa tatattatta tacctaataa gttaagcaat gcagggactttttattttgc 7457 attctttttt cccgagtttt atattaaaag attcaataag aataatactgatctttcctc 7517 gttaggagac acatctttta aacgacttat gagtcgacca catataccaaactatgttta 7577 caatcttgtg ataaactcta atggatgtac ttttaactcc attaaattattattgctggc 7637 tcttagtcta acatcaaaaa ggttttatga aacacctcag caagaacgtaattttttgtg 7697 tcatataaat gaaattgtct tggctaatgc tgacgaatac tccggtatcatttcttgcat 7757 tataaaaagt agaatatctg taattgatga ctttatttca agtaatgtttcattaaatac 7817 aaacaggcaa atagctttat tcataactgg acaatcaaga ggttttatagatgccctacc 7877 taatctcgta agtgaaataa cgattccttc tgatgttgat gtttttattagtacatggaa 7937 agatatcgga cacacacagt tatctaaaga aagaatatgt aggatatttgacagcgaggc 7997 tgcacaatat gtttcagagc cagataacta ctcgtttgta gacgaacactatgatgaatt 8057 aaaagacttg agcttaagtt catataaaaa taataattta gaggagatatattcaagttt 8117 tttctctgga tgtaactcag ttttaataaa cattaaagat gatggggaatatccatataa 8177 taaaatgagt aatgcagaaa aaatgtatta ccataattca ttttggttctgtagtcttaa 8237 aaatcataat tgggataaat ataggtgcat tataaagata aggcctgatgctttattgca 8297 agtggataat gtgacaatta atgatataga tgtagatgat tctgtttattgtgaggatag 8357 taatgggtgg atatttagag aatgggggtt tggcataggc gaccaattattttacggcga 8417 tcctgatata atgaaaaagc tgatgtgtgt ccatggttta gaaaaaatatatagtcagct 8477 aacatccttg atctcaagtt ctaatgttta ttattcaggt catattaatgtagggttatg 8537 tgcttgggct aatgtatatg attgccaggt ttctaattta aaaataaaaaatattgttag 8597 cgcctcgaaa aatatcgcta gaacaaatac tttctttgcg ggaatgagttatttattgca 8657 aggtatagat atcacttaac aatgaaagat gcactatatg aaaaaaataattgtagattt 8717 agataacacc atatctttta atttatcagg aaaatattca catgcaactccaaacaaaaa 8777 actaattgaa aaattgtatg aatataagct taatggtttt tatattgttatttttacagc 8837 aaggaatatg aggacatata aagaaaatat aggtaagatt aatattcatacattaccagt 8897 tataattgat tggttgaatg aaaatagagt cccttatgat gaggtgattgttggtaaacc 8957 ttggtgtgga gatgaggggt tttatgttga tgatagagct attcgaccatcagaactttg 9017 caatatgacc ttagaagaga tttctaatat gttagaacag gagaaaaaatgcttctaata 9077 atgtctggtt cctatgttca acaagaatta ggggccgaat ttggttctattcctccaagc 9137 tttcttcctt tagctaataa acgattattt aagcatcaag tatctttagggcatgatggt 9197 catgcaatat atctggtttt accggaagat tttgtgtttg acaaacatgattatgaatgg 9257 ttgcttcgta ataaagtaac aatgatccct gtcgatagta acttgacattagggcaagcg 9317 atagttaccg catggaattt aataggagat aaagatgaca aaggcttacaattattgttt 9377 ggcgatacac tctttaaaaa aattcctgca ggggaggaat tagtagcaaaaagtcatcct 9437 gatgaaaatt atcaatgggc cattttttac gaaacagagt taagagccgtcagtagagaa 9497 gataataaaa atgtaatttg tgggtatttt tcttttagaa aaccgaatttttttattagg 9557 gaattagtta cttcaaaatt tgattttacg gcggcactta aaaagtatcacgacagctat 9617 agtttagcct ctatatacgt gtctgattgg cttgattttg gacatattaatacatactat 9677 aagtcaaaag tacaatacac aacccagcgt gcatttaatg aattatgcattacaacaaaa 9737 tccgttatca aatcaagttc aaatgaaagt aaaattgaag ctgaatcaaaatggtttgaa 9797 actattcccg gagaattaaa gatctatact ccaatgttat tggaaccgtttgatcatatc 9857 agaaagagtt ataagcttga atatttatat aatacgacgt taaatgaattatttgttttt 9917 tctcgcctac caaataatat tttaacaaat atattaataa gttgtttagacttcatcgat 9977 ctgtgcaaag aatatcattc aattgatact gacaaaaata tactgcaagatttattttat 10037 gaaaaaacga ttgagcgggt tagcaagtac ataacagatt taaatattgatccaaatgca 10097 aaatggaatt ttaataataa tataagcgtt tcaattaatg atattctttatgatactaat 10157 aaatttatcc caagtgaact gcaatataaa actattatgc atggcgatttatgctttagt 10217 aatataattt ttaactttag aactggtaga atacaagttt ttgatcccagaggattgaac 10277 cactctggag aaataagtat ttatggtgat tttcgttatg atatagctaaattatcacat 10337 tcaatactag ggctctatga ttggataatt gcaggatatt atataataaataaaaaaaat 10397 aaaactcata gtattgaatt caaaattaat attgataata aattgtttgaaattcaatca 10457 acatttgttt ctataataaa agagaaatat tcaatctccg aaaaatcattgtatgcgatg 10517 caaatacatt tatttttatc aatgcttccc cttcattccg atgacaaaaaaaggcaagat 10577 gcactatttg ctaatgcatt tagattatat gaaattttta aggaggctgcagtatgatta 10637 taattccaat ggcggggatg agttcgcgtt ttttcaaggc tggatattccaaaccaaaat 10697 atatgcttga attgaatggt gagtttctat tcgatctatg tttgaaaagtttcaaattat 10757 attttgagac tgaacatttt gtctttatcc ttagggatgt tttcaatacgaagtcttttg 10817 tattacaaag aatagcatct ttagggatta atagctacac cttgattactcttgataaag 10877 aaactcgggg gcaagcagaa acagtatatt tggctatatc aaaattatttaatatagaac 10937 aaccaatcac tatttttaac attgatacaa ttaggcctaa ttttatatttactaagttcg 10997 aaggggaaaa tgaatgttat attgaagtat ttcgaggaga tggggataactggtcttttg 11057 ttatgccatc aaatgatgta aaaaatgagg tcattgctac tagcgaaaaaaaacaaattt 11117 ctaacttatg ctgcacagga ttatatcatt tttctacaat taaaaattttatttcagcat 11177 atgaacatta taaaaatcta cctcaagaaa attgggatgc tgggagagttatatatagcc 11237 ccaatataca attatctaat tagtaatggg atcaaagtgt attatacagaaataaataag 11297 tctgatgtta ttttttgtgg tactcctaga gaatatgaaa atttgcaaggaaaaaaataa 11357 aaattaggtt tggcctaata aatctgataa tttattgtta tcaatgcttaatcacatttc 11417 tttgatttta tacacggaat taaatataat ctattatgaa aattgcagttgctggtgtag 11477 gatatgttgg tatatcaatt gctatattac tttcacaaaa acatgatattatcgctctcg 11537 atatagatcc taagaaagtt cagttgatta ataaaaaaat atcaccaatatgtgatcctg 11597 aaatacaaaa atttttatct aatagaaaat taaacctata tgctacaacagaaaaatacg 11657 aagcgtatag agatgctgat tatgttataa tcgcaacacc aaccaattatgatcccatta 11717 ataataactt cgatacactc tcagtagaat cagtagcatg tgacgtactaagtataaatc 11777 ctaatgcaac tatcataatt aaatctacag tccccgtcgg atttactgaacgactaaaac 11837 gcgatctaaa cacgaataat attatctttt ccccagaatt tttacgtgaaggtaaagctc 11897 tttatgacaa cctatatcca tctcgtatag ttgtgggaga gagtagcgaacgagcaagaa 11957 agttcgcaga gcttctcagt gaaggcgcta taaaaaaaga tattccaatattgttaacgg 12017 atagccctga agctgaagcc attaaacttt ttgcaaatac ttaccttgcaatgcggattg 12077 cttatttcaa tgaattggat acttatgcct ccgttcatgg tttagatacaaagcaaatta 12137 tagagggtgt tagtttagat cctagaattg gtcaacatta taataatccttcttttggtt 12197 atggaggtta ctgcttacct aaggatacca agcaatcact cgcaaattatcgtgatgttc 12257 cgcagaactt aatccaggct attgtcgatg ccaatactac ccgaaaagactttgttgcgg 12317 aggatatatt aagtcgtaaa ccaaaagttg taggaatcta tcgcctcataatgaaagcag 12377 gtagtgataa ctttagagca agtagtattc aaggtgtaat gaaacgactcaaagccaaag 12437 gaattgagat agttgtatat gaacctgtac taaaagagcc ttatttctttggttcttatg 12497 ttgagcgtga tattaattct tttaaagaac gtgttgatgt tatagtagccaatcgccgca 12557 cgtcagaatt agaagatgta agtgaaaaag tttatacgcg agatttatttggtgtcgact 12617 cttgattatg ttcaataatt taaaattttt atggctatta aagaaaagtcgatatgtaca 12677 tgctttagct gcaatacaag atgactgccg attttggcag tcaaaacgtatattggcaat 12737 gtacaggctt aatatgtatt ggtcattaca taatcttact gatacaccgtcagattggcg 12797 gtgtaaatta gcaatcaaaa tagctaaaat tgcctgtggt gacataagcttaactccgga 12857 attactcatg gagtttaaag acgagttcac agacacacat caaaaagttgagttagcaaa 12917 aaccttagca tcatactctc ctacgttttc attatcatta ttagataatgtcgataattg 12977 tcccttagac ttgtatcagc tcttcaatta agaatcgggt taactcaaaaagctatatca 13037 acactcgctc agattgatgc cagtgatatt gtatattccc ctgatatattactcttgcaa 13097 aaataatgct ttcagagaaa cggcagaaat ctcgttaaat agacttaacgaatactataa 13157 gtactttggt ttatctccgg tcgcactgac agataactca tctcctttgtcaccttgtaa 13217 tattattaca tcgattcctt atcctgccca aacgggcccc ctgatttctattttaatgac 13277 aacatacaat accggtaggc gggtagaaaa tgcagtaata tcattgcttaatcaaacata 13337 ccgttcattt gagctaatta ttgtggatga tgccagcacc gatgatacgctattccgtct 13397 tcagagatta gcactcaaag atactcgaat aaaaattatt agcctgccacaaaatgttgg 13457 aacatatgct gcaaaacgaa taggcttaat acaggcaaag ggagagtttgtgacatgcca 13517 tgactcagat gactggtccc atcctgaaaa attatttaga cagatatcacctttattgtt 13577 aaaccctaaa ctaatttgtt cgatttctga ttgggtaagg ttgcaagataatgggatttt 13637 ctatgcgcgt gcggtctatc cactaaaaag actgaatcct tcttctctgttgtttagaag 13697 agcggatgta ttgcaaaaag cgggcgtttg ggactgtgtt aaaacgggggctgatagtga 13757 attcattgct cgacttaagc taatttttgg tgattccact gtacatcgtattaaattgcc 13817 tttgacgcta ggaagccatc gtaccgactc gttaatgaat tcacctacaacaggatatac 13877 atctcaggga atttcaccag atcgccaaaa atattgggat tcctggtcgcgatggcacat 13937 tcaggcgtta agaaataaag aaagtcttta cataggaaat tctgatttcactaataaaaa 13997 tcgaccattt tctgcgcccg actcaatatt agtagatact aatgccatcaaaactgcatt 14057 acaaagtgct catgttaact ttaccagtat ataacttatc actaaatgtataatctataa 14117 tatttatttt aataatttat tgtgttttct aattatagta tgttaatcatttatttaatg 14177 aatgggagtt tatgaatggt tatgttaatg ccattcattg tgatgtttttttgatagcat 14237 aatacaatct tttttatctt tttttatatt tttttatctt gttaaaaatcatctccccat 14297 aataaaccgc attaacctgc gtcttaacca ataaataccc tcgaaacttcttaaacagtt 14357 tcatattcgg tttaaaatcg gcctgccaga actggtgcaa atgcccttgatacgtcaagc 14417 ctttgatgtc gtacagggca ttgcccatca ctttgagtgg tttgttatgaatcagcgcag 14477 agatcc 14483 4 686 PRT Escherichia coli 4 Met Ser IleLeu Asn Gln Ala Ile Asn Leu Tyr Lys Asn Lys Asn Tyr 1 5 10 15 Arg GlnAla Leu Ser Leu Phe Glu Lys Val Ala Glu Ile Tyr Asp Val 20 25 30 Ser TrpVal Glu Ala Asn Ile Lys Leu Cys Gln Thr Ala Leu Asn Leu 35 40 45 Ser GluGlu Val Asp Lys Leu Asn Arg Lys Ala Val Ile Asp Ile Asp 50 55 60 Ala AlaThr Lys Ile Met Cys Ser Asn Ala Lys Ala Ile Ser Leu Asn 65 70 75 80 GluVal Glu Lys Asn Glu Ile Ile Ser Lys Tyr Arg Glu Ile Thr Ala 85 90 95 LysLys Ser Glu Arg Ala Glu Leu Lys Glu Val Glu Pro Ile Pro Leu 100 105 110Asp Trp Pro Ser Asp Leu Thr Leu Pro Pro Leu Pro Glu Ser Thr Asn 115 120125 Asp Tyr Val Trp Ala Gly Lys Arg Lys Glu Leu Asp Asp Tyr Pro Arg 130135 140 Lys Gln Leu Ile Ile Asp Gly Leu Ser Ile Val Ile Pro Thr Tyr Asn145 150 155 160 Arg Ala Lys Ile Leu Ala Ile Thr Leu Ala Cys Leu Cys AsnGln Lys 165 170 175 Thr Ile Tyr Asp Tyr Glu Val Ile Val Ala Asp Asp GlySer Lys Glu 180 185 190 Asn Ile Glu Glu Ile Val Arg Glu Phe Glu Ser LeuLeu Asn Ile Lys 195 200 205 Tyr Val Arg Gln Lys Asp Tyr Gly Tyr Gln LeuCys Ala Val Arg Asn 210 215 220 Leu Gly Leu Arg Ala Ala Lys Tyr Asn TyrVal Ala Ile Leu Asp Cys 225 230 235 240 Asp Met Ala Pro Asn Pro Leu TrpVal Gln Ser Tyr Met Glu Leu Leu 245 250 255 Ala Val Asp Asp Asn Val AlaLeu Ile Gly Pro Arg Lys Tyr Ile Asp 260 265 270 Thr Ser Lys His Thr TyrLeu Asp Phe Leu Ser Gln Lys Ser Leu Ile 275 280 285 Asn Glu Ile Pro GluIle Ile Thr Asn Asn Gln Val Ala Gly Lys Val 290 295 300 Glu Gln Asn LysSer Val Asp Trp Arg Ile Glu His Phe Lys Asn Thr 305 310 315 320 Asp AsnLeu Arg Leu Cys Asn Thr Pro Phe Arg Phe Phe Ser Gly Gly 325 330 335 AsnVal Ala Phe Ala Lys Lys Trp Leu Phe Arg Ala Gly Trp Phe Asp 340 345 350Glu Glu Phe Thr His Trp Gly Gly Glu Asp Asn Glu Phe Gly Tyr Arg 355 360365 Leu Tyr Arg Glu Gly Cys Tyr Phe Arg Ser Val Glu Gly Ala Met Ala 370375 380 Tyr His Gln Glu Pro Pro Gly Lys Glu Asn Glu Thr Asp Arg Ala Ala385 390 395 400 Gly Lys Asn Ile Thr Val Gln Leu Leu Gln Gln Lys Val ProTyr Phe 405 410 415 Tyr Arg Lys Lys Glu Lys Ile Glu Ser Ala Thr Leu LysArg Val Pro 420 425 430 Leu Val Ser Ile Tyr Ile Pro Ala Tyr Asn Cys SerLys Tyr Ile Val 435 440 445 Arg Cys Val Glu Ser Ala Leu Asn Gln Thr IleThr Asp Leu Glu Val 450 455 460 Cys Ile Cys Asp Asp Gly Ser Thr Asp AspThr Leu Arg Ile Leu Gln 465 470 475 480 Glu His Tyr Ala Asn His Pro ArgVal Arg Phe Ile Ser Gln Lys Asn 485 490 495 Lys Gly Ile Gly Ser Ala SerAsn Thr Ala Val Arg Leu Cys Arg Gly 500 505 510 Phe Tyr Ile Gly Gln LeuAsp Ser Asp Asp Phe Leu Glu Pro Asp Ala 515 520 525 Val Glu Leu Cys LeuAsp Glu Phe Arg Lys Asp Leu Ser Leu Ala Cys 530 535 540 Val Tyr Thr ThrAsn Arg Asn Ile Asp Arg Glu Gly Asn Leu Ile Ser 545 550 555 560 Asn GlyTyr Asn Trp Pro Ile Tyr Ser Arg Glu Lys Leu Thr Ser Ala 565 570 575 MetIle Cys His His Phe Arg Met Phe Thr Ala Arg Ala Trp Asn Leu 580 585 590Thr Glu Gly Phe Asn Glu Ser Ile Ser Asn Ala Val Asp Tyr Asp Met 595 600605 Tyr Leu Lys Leu Ser Glu Val Gly Pro Phe Lys His Ile Asn Lys Ile 610615 620 Cys Tyr Asn Arg Val Leu His Gly Glu Asn Thr Ser Ile Lys Lys Leu625 630 635 640 Asp Ile Gln Lys Glu Asn His Phe Lys Val Val Asn Glu SerLeu Ser 645 650 655 Arg Leu Gly Ile Lys Lys Tyr Lys Tyr Ser Pro Leu ThrAsn Leu Asn 660 665 670 Glu Cys Arg Lys Tyr Thr Trp Glu Lys Ile Glu AsnAsp Leu 675 680 685 5 27 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 5acccaacact gctacaacct atatcgg 27 6 30 DNA ARTIFICIAL SEQUENCE SYNTHETICDNA 6 gcgtcttcac caataaatac ccacgaaact 30 7 26 DNA ARTIFICIAL SEQUENCESYNTHETIC DNA 7 cgagaaatac gaacacgctt tggtaa 26 8 28 DNA ARTIFICIALSEQUENCE SYNTHETIC DNA 8 actcaatttt ctctttcagc tcttcttg 28 9 30 DNAARTIFICIAL SEQUENCE SYNTHETIC DNA 9 cgggatcccg atgagtattc ttaatcaagc 3010 30 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 10 ggaattccgg ccagtctacatgtttatcac 30 11 108 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 11 atg gggggt tct cat cat cat cat cat cat ggt atg gct agc atg act 48 Met Gly GlySer His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 ggt ggacag caa atg ggt cgg gat ctg tac gac gat gac gat aag gat 96 Gly Gly GlnGln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 cga tgg atcccg 108 Arg Trp Ile Pro 35 12 36 PRT ARTIFICIAL SEQUENCE SYNTHETIC DNA12 Met Gly Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 510 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 2025 30 Arg Trp Ile Pro 35

What is claimed is:
 1. A chondroitin polymerase having the followingproperties: said polymerase transfers GlcUA and GalNAc alternately to anon-reduced terminal of a sugar chain from a GlcUA donor and a GalNAcdonor, respectively; said polymerase transfers GlcUA to anoligosaccharide having GalNAc on its non-reduced terminal and achondroitin backbone from a GlcUA donor, but does not substantiallytransfer GalNAc to the oligosaccharide from a GalNAc donor; saidpolymerase transfers GalNAc to an oligosaccharide having GlcUA on itsnon-reduced terminal and a chondroitin backbone from a GalNAc donor, butdoes not substantially transfer GlcUA to the oligosaccharide from aGlcUA donor; and said polymerase acts in the presence of Mn²⁺ ion butdoes not substantially act in the presence of Ca²⁺ ion, Cu²⁺ ion orethylenediaminetetraacetic acid, wherein GlcUA represents D-glucuronicacid; and GalNAc represents N-acetyl-D-gaiactosamine.
 2. The chondroitinpolymerase according to claim 1, which is derived from Escherichia coli.3. A protein selected from the group consisting of the following (A) and(B): (A) a protein comprising the amino acid sequence represented by SEQID NO: 2; (B) a protein comprising the amino acid sequence in which oneor a few amino acid residue(s) in the amino acid sequence represented bySEQ ID NO: 2 are deleted, substituted, inserted or transposed, andhaving a chondroitin polymerase activity.
 4. A DNA comprising any one ofthe following (a) to (c): (a) a DNA which encodes a protein consistingof the amino acid sequence represented by SEQ ID NO: 2; (b) a DNA whichencodes a protein consisting of an amino acid sequence in which one or afew amino acid residue(s) in the amino acid sequence represented by SEQID NO: 2 are deleted, substituted, inserted or transposed, and having achondroitin polymerase activity; (c) a DNA which hybridizes with (i) theDNA in (a), (ii) a DNA complementary to the DNA in (a), or (iii) a DNAhaving a part of nucleotide sequences of the DNA in (i) and (ii) understringent conditions.
 5. The DNA according to claim 4, wherein the DNAin (a) is represented by SEQ ID NO:
 1. 6. A vector comprising the DNA ofclaim 4 or
 5. 7. The vector according to claim 6, which is an expressionvector.
 8. A transformant in which a host is transformed with the vectorof claim 6 or
 7. 9. A process for producing a chondroitin polymerase,which comprises: growing the transformant of claim 8; and collecting achondroitin polymerase from the grown material.
 10. A sugar chainsynthesizing agent, comprising an enzyme protein which comprises anamino acid sequence represented by the following (A) or (B) and hasenzymatic activities of the following (i) and (ii): (A) the amino acidsequence represented by SEQ ID NO: 2; (B) an amino acid sequence inwhich one or a few amino acid residue(s) in the amino acid sequencerepresented by SEQ ID NO: 2 are deleted, substituted, inserted ortransposed; (i) GlcUA and GalNAc are alternately transferred to anon-reduced terminal of a sugar chain from a GlcUA donor and a GalNAcdonor, respectively; (ii) GlcNAc is transferred to a non-reducedterminal of a sugar chain having GlcUA on the non-reduced terminal froma GlcNAc donor, wherein GlcUA represents D-glucuronic acid; GalNAcrepresents N-acetyl-D-galactosamine; and GlcNAc representsN-acetyl-D-glucosamine.
 11. A process for producing a sugar chainrepresented by the following formula (3), which comprises at least astep of allowing the synthesizing agent of claim 10 to contact with aGalNAc donor and a sugar chain represented by the following formula (1):GlcUA-X—R¹   (1) GalNAc-GlcUA-X—R¹   (3) wherein GlcUA representsD-glucuronic acid; GalNAc represents N-acetyl-D-galactosamine; Xrepresents GalNAc or GlcNAc in which GlcNAc representsN-acetyl-D-glucosamine; - represents a glycosidic linkage; and R¹represents an any group.
 12. A process for producing a sugar chainrepresented by the following formula (4), which comprises at least astep of allowing the synthesizing agent of claim 10 to contact with aGlcNAc donor and a sugar chain represented by the following formula (1):GlcUA-X—R¹   (1) GlcNAc-GlcUA-X—R¹   (4) wherein GlcUA representsD-glucuronic acid; GlcNAc represents N-acetyl-D-glucosamine; Xrepresents GalNAc or GlcNAc in which GalNAc representsN-acetyl-D-galactosamine; - represents a glycosidic linkage; and R¹represents an any group.
 13. A process for producing a sugar chainrepresented by the following formula (5), which comprises at least astep of allowing the synthesizing agent of claim 10 to contact with aGlcUA donor and a sugar chain represented by the following formula (2):GalNAc-GlcUA-R²   (2) GlcUA-GalNAc-GlcUA-R²   (5) wherein GlcUArepresents D-glucuronic acid; GalNAc representsN-acetyl-D-galactosamine; X represents GalNAc or GlcNAc in which GlcNAcrepresents N-acetyl-D-glucosamine; - represents a glycosidic linkage;and R² represents an any group.
 14. A process for producing a sugarchain selected from the following formulae (6) and (8), which comprisesat least a step of allowing the synthesizing agent of claim 10 tocontact with a GalNAc donor, a GlcUA donor and a sugar chain representedby the following formula (1): GlcUA-X—R¹   (1)(GlcUA-GalNAc)n-GlcUA-X—R¹   (6) GalNAc-(GlcUA-GalNAc)n-GlcUA-X—R¹   (8)wherein GlcUA represents D-glucuronic acid; GalNAc representsN-acetyl-D-galactosamine; X represents GalNAc or GlcNAc in which GlcNAcrepresents N-acetyl-D-glucosamine; - represents a glycosidic linkage; R¹represents an any group; and n is an integer of 1 or more.
 15. A processfor producing a sugar chain selected from the following formulae (7) and(9), which comprises at least a step of allowing the synthesizing agentof claim 10 to contact with a GalNAc donor, a GlcUA donor and a sugarchain represented by the following formula (2): GalNAc-GlcUA-R²   (2)(GalNAc-GlcUA)n-GalNAc-GlcUA-R²   (7)GlcUA-(GalNAc-GlcUA)n-GalNAc-GlcUA-R²   (9) wherein GlcUA representsD-glucuronic acid; GalNAc represents N-acetyl-D-galactosamine; -represents a glycosidic linkage; R² represents an any group; and nrepresents an integer of 1 or more.
 16. A hybridization probe comprisinga nucleotide sequence complementary to the nucleotide sequencerepresented by SEQ ID NO: 1 or a part thereof.
 17. A glycosyltransfercatalyst which comprises an enzyme protein comprising an amino acidsequence selected from the following (A) and (B), and is capable oftransferring GlcUA, GalNAc and GlcNAc to a non-reduced terminal of asugar chain from a GlcUA donor, a GalNAc donor and a GlcNAc donor,respectively; (A) the amino acid sequence represented by SEQ ID NO: 2;(B) an amino acid sequence in which one or a few amino acid residue(s)in the amino acid sequence represented by SEQ ID NO: 2 are deleted,substituted, inserted or transposed, wherein GlcUA representsD-glucuronic acid; GalNAc represents N-acetyl-D-galactosamine; andGlcNAc represents N-acetyl-D-glucosamine.